RNA integrity and quantification

RNA integrity publication:

Procedures for Quality Control of RNA Samples for Use in Quantitative Reverse Transcription PCR
Tania Nolan 1,2 & Stephen Bustin 2,3
1 Sigma Aldrich,Cambridge UK; 2 Eureka Biotechnology, Cambridge UK; 3 Institute of Cell and Molecular Science, Barts and the London Queen Mary’s School of Medicine and Dentistry, London, UK
Essentials of Nucleic Acid Analysis: A Robust Approach (2008) editors: Jacquie T. Keer, Lyndsey Birch

Book chapter 9 introduction - The quality of any scientiﬁc data is directly proportional to that of theoriginal starting samples, or simply ‘garbage in, garbage out’. In most circumstances it is logical to work with the highest quality material possible. However, for some experiments the highest quality possible is still a serious compromise from perfection. The degree to which the standard of input material inﬂuences ﬁnal quantitative reverse transcription polymerase chain reaction (qRT-PCR) data and, potentially, the resulting scientiﬁc conclusion, is outlined in this chapter.

Preservation of fine-needle aspiration specimens for future use in RNA-based molecular testing.
Ladd AC, O'Sullivan-Mejia E, Lea T, Perry J, Dumur CI, Dragoescu E, Garrett CT, Powers CN.
Cancer Cytopathol. 2011 Apr 25;119(2): 103-110
Department of Pathology, School of Medicine, Virginia Commonwealth University, Richmond, Virginia

BACKGROUND: The application of ancillary molecular testing is becoming more important for the diagnosis and classification of disease. The use of fine-needle aspiration (FNA) biopsy as the means of sampling tumors in conjunction with molecular testing could be a powerful combination. FNA is minimally invasive, cost effective, and usually demonstrates accuracy comparable to diagnoses based on excisional biopsies. Quality control (QC) and test validation requirements for development of molecular tests impose a need for access to pre-existing clinical samples. Tissue banking of excisional biopsy specimens is frequently performed at large research institutions, but few have developed protocols for preservation of cytologic specimens. This study aimed to evaluate cryopreservation of FNA specimens as a method of maintaining cellular morphology and ribonucleic acid (RNA) integrity in banked tissues.
METHODS: FNA specimens were obtained from fresh tumor resections, processed by using a cryopreservation protocol, and stored for up to 27 weeks. Upon retrieval, samples were made into slides for morphological evaluation, and RNA was extracted and assessed for integrity by using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif).
RESULTS: Cryopreserved specimens showed good cell morphology and, in many cases, yielded intact RNA. Cases showing moderate or severe RNA degradation could generally be associated with prolonged specimen handling or sampling of necrotic areas.
CONCLUSIONS: FNA specimens can be stored in a manner that maintains cellular morphology and RNA integrity necessary for studies of gene expression. In addition to addressing quality control (QC) and test validation needs, cytology banks will be an invaluable resource for future molecular morphologic and diagnostic research studies.

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection.
Bevilacqua C, Makhzami S, Helbling JC, Defrenaix P, Martin P.
BMC Cell Biol. 2010 Dec 6;11:95.
INRA, UMR1313 Unité Génétique Animale et Biologie Intégrative, équipe Lait, Génome & Santé F-78350 Jouy-en-Josas, France

BACKGROUND: Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems®) system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.

RESULTS: The best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems®) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 μm2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.

CONCLUSIONS: RNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery.

Impact of RNA degradation on gene expression profiling.
Opitz L, Salinas-Riester G, Grade M, Jung K, Jo P, Emons G, Ghadimi BM, Beissbarth T, Gaedcke J.
BMC Med Genomics. 2010 Aug 9;3: 36.

BACKGROUND: Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis.
METHODS: To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time-dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences.
RESULTS: The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5' end.
CONCLUSIONS: Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5' end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings.

RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays.
Koppelkamm A, Vennemann B, Lutz-Bonengel S, Fracasso T, Vennemann M.
Int J Legal Med. 2011 Jul;125(4): 573-580
Institute of Legal Medicine, Freiburg University Medical Center, Albertstrasse 9, 79104, Freiburg, Germany.

Messenger RNA (mRNA) profiling in post-mortem human tissue might reveal information about gene expression at the time point of death or close to it. When working with post-mortem human tissue, one is confronted with a natural RNA degradation caused by several parameters which are not yet fully understood. The aims of the present study were to analyse the influence of impaired RNA integrity on the reliability of quantitative gene expression data and to identify ante- and post-mortem parameters that might lead to reduced RNA integrities in post-mortem human brain, cardiac muscle and skeletal muscle tissues. Furthermore, this study determined the impact of several parameters like type of tissue, age at death, gender and body mass index (BMI), as well as duration of agony, cause of death and post-mortem interval on the RNA integrity. The influence of RNA integrity on the reliability of quantitative gene expression data was analysed by generating degradation profiles for three gene transcripts. Based on the deduced cycle of quantification data, this study shows that reverse transcription quantitative polymerase chain reaction (RT-qPCR) performance is affected by impaired RNA integrity. Depending on the transcript and tissue type, a shift in cycle threshold values of up to two cycles was observed. Determining RNA integrity number of 136 post-mortem samples revealed significantly different RNA qualities among the three tissue types with brain revealing significantly lower integrities compared to skeletal and cardiac muscle. The body mass index was found to influence RNA integrity in skeletal muscle tissue (M. iliopsoas). Samples originating from deceased with a BMI > 25 were of significantly lower integrity compared to samples from normal weight donors. Correct data normalisation was found to partly diminish the effects caused by impaired RNA quality. Nevertheless, it can be concluded that in post-mortem tissue with low RNA integrity numbers, the detection of large differences in gene expression activities might still be possible, whereas small expression differences are prone to misinterpretation due to degradation. Thus, when working with post-mortem samples, we recommend generating degradation profiles for all transcripts of interest in order to reveal detection limits of RT-qPCR assays.

Analysis of Circulating MicroRNA - Pre-analytical and Analytical Challenges.
McDonald JS, Milosevic D, Reddi HV, Grebe SK, Algeciras-Schimnich A.
Clin Chem. 2011 Jun;57(6):833-40. Epub 2011 Apr 12.
Departments of Laboratory Medicine and Pathology

BACKGROUND: There is great interest in circulating microRNAs (miRNAs) as disease biomarkers. Translating promising miRNAs into validated clinical tests requires the characterization of many preanalytical and analytical parameters.

METHODS: miRNAs were extracted from serum and plasma samples of healthy volunteers, and miRNAs known to be present in serum and plasma (miR-15b, miR-16, miR-24, and miR-122) were amplified by reverse-transcription quantitative PCR. Stability and the effects of hemolysis were determined. Assay variation and its components, including the effect of adding control miRNA, were assessed by nested ANOVA.

RESULTS: miRNA concentrations were higher in plasma than in serum. Processing of plasma to remove subcellular/cellular components reduced miRNA concentrations to those of serum. The miRNAs analyzed were stable refrigerated or frozen for up to 72 h and were stable at room temperature for 24 h. Hemolysis increased the apparent concentration of 3 of the miRNAs. The total variability of replicate miRNA concentrations was <2.0-fold, with most of the variability attributable to the extraction process and interassay imprecision. Normalizing results to those of spiked exogenous control miRNAs did not improve this variability.

CONCLUSIONS: Detailed validation of the preanalytical steps affecting miRNA detection and quantification is critical when considering the use of individual miRNAs as clinical biomarkers. Unless these causes of imprecision are considered and mitigated, only miRNAs that are extremely up- or downregulated will be suitable as clinical biomarkers.

Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization.
Antonov J, Goldstein DR, Oberli A, Baltzer A, Pirotta M, Fleischmann A, Altermatt HJ, Jaggi R.
Lab Invest. 2005 Aug;85(8): 1040-1050.
Department of Clinical Research, University of Bern, Bern, Switzerland.

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

Evaluation of different RNA extraction methods for small quantities of plant tissue: Combined effects of reagent type and homogenization procedure on RNA quality-integrity and yield
Mary Portillo, Carmen Fenoll, Carolina Escobar
Physiologia Plantarum (2006) Volume: 128, Issue: 1, Pages: 1-7

Highly sensitive techniques for transcriptome analysis, such as microarrays, complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), and others currently used in functional genomics require a high RNA quality and integrity, as well as reproducibility among extractions of replicates from the same tissue. There are, however, few technical papers comparing different homogenization techniques and reagents to extract RNA from small quantities of plant tissue. We extracted RNA from tomato seedlings with the three different commercial reagents TRIZOL LS, TRIZOL, and TRI Reagent in combination with pulverization, homogenization-maceration in a mortar, and homogenization with mild vibration plus glass beads, and evaluated total RNA integrity-quality and yield. Pulverization under liquid nitrogen combined with TRIZOL LS as extraction reagent and homogenization-maceration in mortar with TRI Reagent, are the procedures that rendered higher RNA yield, integrity and quality, as well as reproducibility among independent RNA extractions. In contrast, short mild vibration pulses (4500r.p.m. for 5s) mixed with glass beads, rendered low extraction efficiency and caused, in most cases, partial RNA degradation.

Combined expression of miR-122a, miR-1, and miR-200b can differentiate degraded RNA samples from liver, pancreas, and stomach.
Kim J, Choi NE, Oh SJ, Park SJ, Kim HK.
Pathol Int. 2011 Feb;61(2): 67-72
National Cancer Center, Goyang, Republic of Korea.

The effect of RNA degradation on the diagnostic utility of microRNA has not been systematically evaluated in clinical samples. We asked if the microRNA profile is preserved in degraded RNA samples derived from mouse and human tissue. We selected tissue-specific microRNA candidates from published human microarray data, and validated them using quantitative reverse transcription polymerase chain reaction (QRTPCR) analyses on flash-frozen, normal mouse liver, pancreas, and stomach tissue samples. MiR-122a, miR-1, and miR-200b were identified as tissue-specific, and the 3-microRNA-based QRTPCR could predict the tissue origin for mouse tissue samples that were left at room temperature for 2 h with an accuracy of 91.7%. When we applied this 3-microRNA predictor to clinical specimens with various degree of RNA degradation, the predictor differentiated degraded RNA samples from liver, pancreas, and stomach with an accuracy of 90% (26/29). Expression levels of miR-122a, miR-1, and miR-200b were modestly changed after the extended (2-4 h) storage at room temperature, but the magnitudes of expression changes were small compared to the expression differences between various tissues of origin. This proof-of-principle study demonstrates that RNA degradation due to extended storage at room temperature does not affect the predictive power of tissue-specific microRNA QRTPCR predictor.

A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue.
Cummings M, McGinley CV, Wilkinson N, Field SL, Duffy SR, Orsi NM.
Anal Biochem. 2011 Sep 1;416(1): 123-125
Gynaeimmunology and Oncology Group, YCR and Liz Dawn Pathology and Translational Sciences Centre, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds LS9 7TF, UK.

Laser capture microdissection of frozen tissue sections allows homogeneous cell populations to be isolated for expression profiling. However, this requires striking a balance between retaining adequate morphology for accurate microdissection and maintaining RNA integrity. Various staining protocols were applied to frozen endometrial carcinoma tissue sections. Although alcohol-based methods were superior to aqueous stains for maintaining RNA integrity, they suffered from irreproducible staining intensity. We developed a modified alcohol-based, buffered cresyl violet staining protocol that provides reproducible staining with minimal RNA degradation suitable for tissues with moderate to high levels of intrinsic RNase activity.

Effects of RBC removal and TRIzol of peripheral blood samples on RNA stability.
Kang JE, Hwang SH, Lee JH, Park DY, Kim HH.
Clin Chim Acta. 2011 Jun 17
Department of Laboratory Medicine, Pusan National University Hospital, School of Medicine, Pusan National University, Busan, South Korea.

BACKGROUND: Purification of mRNA from stored specimens is very important because results from RT-PCR and microarray analyses are largely affected by the quality of mRNA. Moreover, many preanalytical factors during collection, processing, and storage may affect mRNA quality and the expression of peripheral blood mononuclear cells (PBMC). In this study, we evaluate the effects of RBC removal techniques and TRIzol on RNA quality in blood samples.
METHODS: We obtained EDTA-blood samples from 50 adult volunteers, and made 10 pools of buffy coats for comparison between protocols and also evaluated RNA quality of clinical samples in biobank. Use of TRIzol and RBC removal (RBC lysis or cell separation) were evaluated their effect on the quality of mRNA from the stored blood samples.
RESULTS: RNA integrity with TRIzol was significantly better than that without TRIzol (RIN 4.5 vs. 9.2, respectively; P=0.002). The change in RIN of the PBMC separation method was equivalent to that of the RBC lysis method. After 12months, IL6 mRNA expression from stored clinical samples in cell separation/TRIzol was stable.
CONCLUSIONS: The blood samples frozen in TRIzol after RBC removal preserved RNA quality well. PBMC/TRIzol preservation for storage of blood samples could be a simple protocol for rapid, low-cost biobanking.

Effects of delay in the snap freezing of colorectal cancer tissues on the quality of DNA and RNA.
Hong SH, Baek HA, Jang KY, Chung MJ, Moon WS, Kang MJ, Lee DG, Park HS.
J Korean Soc Coloproctol. 2010 Oct;26(5): 316-323
Chonbuk National University Hospital National Biobank of Korea, Jeonju, Korea.

PURPOSE: The success of basic molecular research using biospecimens strongly depends on the quality of the specimen. In this study, we evaluated the effects of delayed freezing time on the stability of DNA and RNA in fresh frozen tissue from patients with colorectal cancer.
METHODS: Tissues were frozen at 10, 30, 60, and 90 minutes after extirpation of colorectal cancer in 20 cases. Absorbance ratio of 260 to 280 nm (A(260)/A(280)) and agarose gel electrophoresis were evaluated. In addition, the RNA integrity number (RIN) was assayed for the analysis of the RNA integrity.
RESULTS: Regardless of delayed freezing time, all DNA and RNA samples revealed A(260)/A(280) ratios of more than 1.9, and all DNA samples showed a discrete, high-molecular-weight band on agarose gel electrophoresis. The RINs were 7.53 ± 2.04, 6.70 ± 1.88, 6.47 ± 2.58, and 4.22 ± 2.34 at 10, 30, 60, and 90 minutes, respectively. Though the concentration of RNA was not affected by delayed freezing, the RNA integrity was decreased with increasing delayed freezing time.
CONCLUSION: According to the RIN results, we recommend that the collection of colorectal cancer tissue should be done within 10 minutes for studies requiring RNA of high quality and within 30 minutes for usual RNA studies.

Improved protocol for high-quality co-extraction of DNA and RNA from rumen digesta.
Popova M, Martin C, Morgavi DP.
Folia Microbiol (Praha). 2010 Jul;55(4): 368-372
INRA Clermont-Ferrand-Theix, 63122, Saint Gènes Champanelle, France.

We report an improved method for total nucleic acids extraction from rumen content samples. The method employs bead beating, and phenol-chloroform extraction followed by saline-alcohol precipitation. Total nucleic acids and RNA yield and purity were assessed by spectrophotometric measurements; RNA integrity was estimated using Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method provided total nucleic acids and RNA extracts of good quantity and quality. The extraction is not time consuming and it is valuable for ecological studies of rumen microbial community structure and gene expression.

mRNA profiling in forensic genetics I - Possibilities and limitations.
Vennemann M & Koppelkamm A.
Institute of Legal Medicine, University of Freiburg, Albertstr. 9, 79104 Freiburg, Germany
Forensic Sci Int. 2010 Dec 15;203(1-3): 71-75

Molecular investigations gain increasing interest in forensic medicine. Examination of gene expression levels at the time point of death might have the power to become a complementing tool to the current methods for the determination of cause and circumstances of death. This includes pathophysiological conditions of disease and injury as well as the duration of agony or other premortem factors. Additionally, recent developments in forensic genetics revealed that tissue specific mRNAs can be used to determine the type of body fluid present in a crime scene stain. Although RNA is known to be rather instable, RNA could be extracted in adequate quality from tissue samples collected during medico-legal autopsy. Nevertheless, working with human postmortem tissue means to deal with highly variable RNA integrities. This review aims to give a brief overview of the possible advantages of postmortem mRNA profiling and to shed further light into the limitations of this method arising from reduced RNA integrities.

Postmortem mRNA profiling II - Practical considerations.
Vennemann M & Koppelkamm A.
Institute of Legal Medicine, University of Freiburg, Albertstr. 9, 79104 Freiburg, Germany
Forensic Sci Int. 2010 Dec 15;203(1-3): 76-82

Using human postmortem tissues for gene expression studies is particularly challenging. Besides the problem of impaired RNA one has to face a very high degree of biological variance within a sample set. Variations of individual parameters like age, body mass, health, but also the cause and circumstances of death and the postmortem interval lead to a rather inhomogeneous collection of samples. To meet these problems it is necessary to consider certain precautions before starting a gene expression project. These precautions include the sample collection and the determination of the RNA integrity, the number of replicates needed and the methods used for reverse transcription and quantitative polymerase chain reaction, but also the strategy for data normalisation and data interpretation. In this article practical issues are discussed to address some of the problems occurring in the work with postmortem human samples obtained during medico-legal autopsy.

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection
Claudia Bevilacqua email, Samira Makhzami email, Jean-Christophe Helbling email, Pierre Defrenaix email and Patrice Martin email
BMC Cell Biology 2010, Published: December 2010

Background - Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.

Results - The best results were obtained when staining 8 um-thick sections with Cresyl violet(Ambion) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 um^2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (alpha-lactalbumin) and CSN1S2 (alpha s2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.

Conclusions - RNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery.

Robust microRNA stability in degraded RNA preparations from human tissue and cell samples.
Jung M, Schaefer A, Steiner I, Kempkensteffen C, Stephan C, Erbersdobler A, Jung K.
Department of Urology, University Hospital Charité, Berlin, Germany.
Clin Chem. 2010 Jun;56(6): 998-1006.

BACKGROUND: RNA integrity is the essential factor that determines the accuracy of mRNA transcript measurements obtained with quantitative real-time reverse-transcription PCR (RT-qPCR), but evidence is clearly lacking on whether this conclusion also applies to microRNAs (miRNAs). We evaluated this issue by comparative analysis of the dependence of miRNA and mRNA measurements on RNA integrity in renal and prostate samples, under both model and clinical conditions.

METHODS: Samples of total RNA isolated from human renal tissue and Caki-2 cells, as well as from prostate tissue and LNCaP cells, were incubated at 80 degrees C for 5-240 min. We subsequently determined the RNA integrity number (RIN) and used RT-qPCR to measure various miRNAs (miR-141, miR-155, miR-200c, and miR-210 in renal samples, and miR-96, miR-130b, miR-149, miR-205, and miR-222 in prostate samples). We similarly measured mRNAs encoded by CDH16 (cadherin 16, KSP-cadherin), PPIA [peptidylprolyl isomerase A (cycophilin A)], and TBP (TATA box binding protein) in renal samples, and HIF1A [hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)], HPRT1 (hypoxanthine phosphoribosyltransferase 1), and KLK3 (kallikrein-related peptidase 3; also known as PSA) in prostate samples. Additionally, we quantified selected miRNAs and mRNAs in samples of RNAs with different RIN values that we isolated from clinical samples. The effect of RIN on the miRNA and mRNA data was assessed by linear regression analysis and group comparison.

RESULTS: The heat-incubation experiments of cell line and tissue RNAs showed that RIN values had negligible or no effect on miRNA results, whereas all mRNAs gradually decreased with decreasing RIN values. These findings were corroborated by our findings with clinical samples.

CONCLUSIONS: Our results suggest the stability of miRNAs to be generally robust, which makes feasible accurate miRNA measurements with RT-qPCR, even in degraded RNA preparations for which reliable mRNA analyses are commonly inapplicable.

Assessment of mRNA and microRNA stabilization in peripheral Human Blood for Multicenter studies and Biobanks
Daniel Gilbert Weber1, Swaantje Casjens1, Peter Rozynek1, Martin Lehnert1, Sandra Zilch-Schöneweis1, Oleksandr Bryk1, Dirk Taeger1, Maria Gomolka2, Michaela Kreuzer2, Heinz Otten3, Beate Pesch1, Georg Johnen1 and Thomas Brüning11
Institute for Prevention and Occupational Medicine of the German Social Accident Insurance—Institute of the Ruhr-Universität Bochum (IPA), Bochum, Germany. 2Department of Radiation Protection and Health, Federal Office for Radiation Protection, Oberschleissheim, Germany. 3German Social Accident Insurance (DGUV), Sankt Augustin, Germany.
Biomarker Insights 2010(5): 95–102

In this study we evaluate the suitability of two methods of RNA conservation in blood samples, PAXgene and RNAlater, in combination with variable shipping conditions for their application in multicenter studies and biobanking. RNA yield, integrity, and purity as well as levels of selected mRNA and microRNA species were analyzed in peripheral human blood samples stabilized by PAXgene or RNAlater and shipped on dry ice or at ambient temperatures from the study centers to the central analysis laboratory. Both examined systems were clearly appropriate for RNA stabilization in human blood independently of the shipping conditions. The isolated RNA is characterized by good quantity and quality and well suited for downstream applications like quantitative RT-PCR analysis of mRNA and microRNA. Superior yield and integrity values were received using RNAlater. It would be reasonable to consider the pro-duction and approval of blood collection tubes prefilled with RNAlater to facilitate the use of this excellent RNA stabilization system in large studies.

Pre-PCR Processing - Strategies to Generate PCR-Compatible Samples
Peter Rådström,* Rickard Knutsson, Petra Wolffs,Maria Lövenklev, and Charlotta Löfström
MOLECULAR BIOTECHNOLOGY Volume 26, 2004: 133-146

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostictool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dra-matically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reducethe effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products,that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCRprocessing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use ofalternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparationmethod, (3) optimization of the sampling method, and (4) combinations of the different strategies. Thisreview describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate andprecise DNA amplification.

COMPARISON OF TWO AVAILABLE PLATFORMS FOR DETERMINATION OF RNA QUALITY
I. Riedmaier, M. Bergmaier and M.W. Pfaffl
Technische Universitat Munchen, Physiology Weihenstephan, Freising, Germany
Biotechnol. & Biotechnol. Equipment 2010, 24(4): 2154-2159

The integrity of RNA is a very critical aspect regarding downstream RNA based quantitative analysis like RT-qPCR. Low-quality RNA can compromise the results of such experiments. Today automated lab-on-chip capillary electrophoresis allows rapid RNA quality and quantity determination, e.g. 2100 Bioanalyzer (Agilent Technologies) and the Experion (Bio-Rad). Both platforms determine RNA quality using a numerical system which represents the integrity of RNA. The Bioanalyzer offers the RIN algorithm (RNA Integrity Number) on the Bioanalyzer 2100 and Bio-Rad developed a new Experion software version that offers an algorithm for calculating the RNA Quality Index (RQI).The aim of this study was to compare both systems regarding sensitivity, reproducibility, linearity and the influence of individual tissue extractions and different chip runs on RNA quality and quantity determination.Overall it was confirmed that both algorithms are very comparable and beneficial for the determination of RNA quality for downstream applications. The Experion showed slightly better results regarding reproducibility and absolute sensitivity, whereas the 2100 Bioanalyzer showed a higher linearity.

Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA.
Okello JB, Rodriguez L, Poinar D, Bos K, Okwi AL, Bimenya GS, Sewankambo NK, Henry KR, Kuch M, Poinar HN.
Department of Anthropology, McMaster Ancient DNA Centre, McMaster University, Hamilton, Ontario, Canada. PLoS One. 2010 Nov 10;5(11):e13931.

BACKGROUND: The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues.

METHODOLOGY/PRINCIPAL FINDINGS: We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays.

CONCLUSIONS/SIGNIFICANCE: We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation.

Stabilizing RNA at room temperature in RNAstable
Sharron Ohgi1, Laurent Coulon, Rolf Muller, Judy-Muller-Cohn, and Omoshile ClementBiomatrica, Inc., 5627 Oberlin Dr, #120, San Diego, CA 92121, USA.
Biotechniques Vol. 48 (No. 6) 2010: 470

RNAstable is a novel preservation product developed to protect RNA from degradation during storage or shipment at ambient temperatures. The synthetic storage medium is based on the natural principles of anhydrobiosis (meaning “life without water”), a biological mechanism employed by some organisms that enables their survival while dry for more than 100 years. Anhyd-robiotic organisms protect their DNA, RNA, proteins, membranes and cellular systems for survival in a dry state and can be revived by simple rehydration. RNAstable was designed to mimic these unique characteristics to stabilize RNA at ambient tempera-tures for prolonged time periods. Quantitative RT-PCR analysis demonstrates successful amplification of RNA templates that were stored dry in RNAstable for 29 months at room temper-ature and under accelerated aging conditions equivalent to 12 years of room temperature storage (elevated temperatures at 45°C). Samples were sealed inside a moisture-barrier bag including a desiccant pack to ensure ideal storage conditions. Rehydrated samples were used directly in reactions without further purification and exhibited no inhibition or loss of activity. This innovative technology prevents degradation of RNA at room temperature and offers tremendous cost and energy savings as an easy-to-use alternative to conventional freezer storage.

A guide to ions and RNA structure.
Draper DE.
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA
RNA. 2004 Mar;10(3):335-43.

RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. "Diffuse ions" accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A "chelated" ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg(2+) on RNA stability where experimental data are available. An important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.

DNA integrity publication:

Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.
Didelot A, Kotsopoulos SK, Lupo A, Pekin D, Li X, Atochin I, Srinivasan P, Zhong Q, Olson J, Link DR, Laurent-Puig P, Blons H, Hutchison JB, Taly V.
Université Paris Sorbonne Cité, INSERM UMR-S775, Paris, France.
Clin Chem. 2013 May;59(5): 815-823

BACKGROUND: Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA.
METHODS: Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues.
RESULTS: One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants.
CONCLUSIONS: The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.

Incorporation of measurement of DNA integrity into qPCR assays.
Brisco M, Latham S, Bartley P, Morley A.
Biotechniques. 2010 Dec;49(6): 893-897.
Department of Haematology and Genetic Pathology, Flinders University and Medical Centre, Bedford Park, South Australia, Australia.

Optimal accuracy of quantitative PCR (qPCR) requires correction for integrity of the target sequence. Here we combine the mathematics of the Poisson distribution and exponential amplification to show that the frequency of lesions per base (which prevent PCR amplification) can be derived from the slope of the regression line between cycle threshold (Ct) and amplicon length. We found that the amplifiable fraction (AF) of a target can be determined from this frequency and the target length. Experimental results from this method correlated with both the magnitude of a damaging agent and with other measures of DNA damage. Applying the method to a reference sequence, we determined the values for lesions/base in control samples, as well as in the AFs of the target sequence in qPCR samples collected from leukemic patients. The AFs used to calculate the final qPCR result were generally >0.5, but were <0.2 in a few samples, indicating significant degradation. We conclude that DNA damage is not always predictable; quantifying the DNA integrity of a sample and determining the AF of a specific qPCR target will improve the accuracy of qPCR and aid in the interpretation of negative results.

Implications of storing urinary DNA from different populations for molecular analyses.
Cannas A, Kalunga G, Green C, Calvo L, Katemangwe P, Reither K, Perkins MD, Maboko L, Hoelscher M, Talbot EA, Mwaba P, Zumla AI, Girardi E, Huggett JF; TB trDNA consortium.
National Institute for Infectious Diseases L. Spallanzani, IRCCS, Roma, Italy.
PLoS One. 2009 Sep 10;4(9): e6985.

BACKGROUND: Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days.

METHODOLOGY: Urine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4 degrees C, -20 degrees C & -80 degrees C) with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J) and single copy (TLR2) targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.

CONCLUSION: Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.

Method for isolation of PCR-ready genomic DNA from zebrafish tissues.
Meeker ND, Hutchinson SA, Ho L, Trede NS.
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.
Biotechniques. 2007 Nov;43(5):610, 612, 614.

Here we describe a method for the isolation of PCR-ready genomic DNA from various zebrafish tissues that is based on a previously published murine protocol. The DNA solutions are of sufficient quality to allow PCR detection of transgenes from all commonly used zebrafish tissues. In sperm, transgene amplification was successful even when diluted 1000-fold, allowing easy identification of transgenic founders. Given its speed and low cost, we anticipate that the adoption of this method will streamline DNA isolation for zebrafish research.

Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in tissue from a Danish population undergoing vascular repair

TinaMygind*1, LarsØstergaard2, SvendBirkelund1, JesSLindholt3 and GunnaChristiansen1
1Department of Medical Microbiology and Immunology, Wilhelm Meyers Allé, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark, 2Research Unit Q, Department of Infectious Diseases, Skejby Hospital, University H
BMC Microbiology 2003, 3:19

Background: To date PCR detection of Chlamydia pneumoniae DNA in atherosclerotic lesions from Danish patients has been unsuccessful. To establish whether non-detection was caused by asuboptimal DNA extraction method, we tested five different DNA extraction methods forpurification of DNA from atherosclerotic tissue.Results: The five different DNA extraction methods were tested on homogenate ofatherosclerotic tissue spiked with C. pneumoniae DNA or EB, on pure C. pneumoniae DNA samplesand on whole C. pneumoniae EB. Recovery of DNA was measured with a C. pneumoniae-specificquantitative real-time PCR. A DNA extraction method based on DNA-binding to spin columnswith a silica-gel membrane (DNeasy Tissue kit) showed the highest recovery rate for the tissuesamples and pure DNA samples. However, an automated extraction method based on magneticglass particles (MagNA Pure) performed best on intact EB and atherosclerotic tissue spiked withEB. The DNeasy Tissue kit and MagNA Pure methods and the highly sensitive real-time PCR weresubsequently used on 78 atherosclerotic tissue samples from Danish patients undergoing vascularrepair. None of the samples were positive for C. pneumoniae DNA. The atherosclerotic sampleswere tested for inhibition by spiking with two different, known amounts of C. pneumoniae DNA andno samples showed inhibition.Conclusion: As a highly sensitive PCR method and an optimised DNA extraction method wereused, non-detection in atherosclerotic tissue from the Danish population was probably not causedby use of inappropriate methods. However, more samples may need to be analysed per patient tobe completely certain on this. Possible methodological and epidemiological reasons for non-detection of C. pneumoniae DNA in atherosclerotic tissue from the Danish population are discussed. Further testing of DNA extraction methods is needed as this study has shownconsiderable intra- and inter-method variation in DNA recovery.

Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues.
Okello JB, Zurek J, Devault AM, Kuch M, Okwi AL, Sewankambo NK, Bimenya GS, Poinar D, Poinar HN.
McMaster Ancient DNA Centre, Department of Anthropology, McMaster University, Hamilton, Ontario L8S4L9, Canada
Anal Biochem. 2010 May 1;400(1): 110-117

Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGen's WaxFree method and our in-house phenol-chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambion's RecoverAll method recovered the most amplifiable RNA.