mRNA Transcript Analysis: Reverse Transcription (3)
mRNA Transcript Analysis: Reverse Transcription (1)
mRNA Transcript Analysis: Reverse Transcription (2)
MOLECULAR BIOLOGY: RNA lost in translation.
Nature Vol 440 23 March 2006
In any manufacturing process, quality control is crucial, and gene expression is no exception. A new pathway monitors mRNAs - the intermediaries of gene expression - and destroys faulty molecules.
Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA:
Michael Stangegaard, Inge Høgh Dufva, and Martin Dufva
Reverse transcription of RNA is an invaluable method for gene expression analysis by real-time PCR or microarray methods. Random primers of varying lengths were compared with respect to their efficiency of priming reverse transcription reactions. The results showed that 15-nucleotide-long random oligonucleotides (pentadecamers) consistently yielded at least 2-fold as much cDNA as did random hexamers using either poly(A) RNA or an amplified version of messenger RNA (aRNA) as a template. The cDNA generated using pentadecamers did not differ in size distribution or the amount of incorporated label compared with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of >80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage of the transcriptome when using random pentadecamers over random hexamers. Using the same amount of aRNA as starting material, random pentadecamer-primed reactions resulted in 11-fold more genes being detected in whole transcriptome DNA microarray experiments than random hexamer-primed reactions. The results indicate that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality.
K. Swinson, M. Koban
Laboratory of Physiology, Richard N. Dixon Science Research Building, Department of Biology, Morgan State University, 1700 E. Cold Spring Lane, Baltimore, Maryland 21251, USA
The labile nature of RNA demands careful procedures for its extraction, purification, and storage. Generally, RNA is solubilized in aqueous buffers or organic solvents, or precipitated with alcohol and then kept at 20 8C or colder. A commercially available product for RNA storage is FORMAzol (Molecular Research Center). We began using FORMAzol because the Application Notes from the Product Description sheet claims that reverse-transcription (RT) is not inhibited so long as FORMAzol does not exceed 5% (v/v) in the reaction mix. This is ostensibly more convenient than having to precipitate RNA, resolubilizing it in water or buffer, and then proceeding with RTPCR. However, amplicon yields for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were poor when using RNA directly from FORMAzol, even though its final concentration was typically much less than 5%. By contrast, satisfactory RT-PCR products were obtained with RNA stored frozen in water or that had been resolubilized from alcohol precipitates. When RT-PCR was then performed on ethanol-precipitated and resolubilized RNA from FORMAzol, yields of GAPDH amplicons were acceptable. Although a revision to the FORMAzol Product Description sheet is now available at the manufacturer’s website (http://www.mrcgene.com/formazol.htm), if users of the product implicitly follow the directions found in the package insert sheet—not being aware of the inhibitory effects of formamide (the denaturant in FORMAzol) — unsatisfactory results may be obtained from RT-PCR experiments. It is suggested that FORMAzol only be used for RNA storage and that RNA be precipitated with alcohol, washed, and resolubilized prior to use.<>
Michael J. Wacker and Michael P. Godard
Applied Physiology Laboratory, University of Kansas, Lawrence, Kansas
Real-time reverse transcription polymerase chain reaction (RT-PCR) is a commonly used technique to analyze gene expression. There has been little research conducted to test if Super-Script III quantitative one-step (reverse transcription carried out in the same tube as PCR) and two-step (reverse transcription carried out in a separate reaction) RT-PCR systems provide similar real-time results. In this study, real-time reactions were set up using the housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), β2-microglobulin (B2M), and RNA polymerase 2 subunit A (PolR2A). Reaction efficiencies were determined by generating standard curves using total RNA isolated from human skeletal muscle and brain. Reaction efficiencies ranged from 97.7 ± 0.9% to 99.4 ± 1.8% for one-step and 98.0 ± 0.2% to 102.6 ± 1.3% for two-step RT-PCR (R2 values for all reactions ≥ 0.995). The sensitivities of one-step and two-step methods, as measured by cycle threshold values, were similar for GAPDH and B2M. However, for the lesser expressed PolR2A mRNA there was a 5 cycle lower threshold for one-step. In summary, both Super-Script III one-step and two-step methods yield reaction efficiencies close to 100% and produce similar, accurate, linear standard curves. However, using the one-step method with gene-specific priming may be more sensitive for quantification of certain genes such as PolR2A.
Reduced Plasma RNA Integrity in Nasopharyngeal Carcinoma Patients
Blenda C.K. Wong, K.C. Allen Chan, Anthony T.C. Chan, Sing-Fai Leung, LisaY.S. Chan, Katherine C.K. Chow, and Y.M. Dennis Lo
Purpose: Recent research has shown the feasibility of detecting cell-free RNA markers in human subjects. As elevated RNase activity has previously been described in the circulation of cancer patients, we hypothesized that cancer patients may have reduced plasma RNA integrity. In this study, we used nasopharyngeal carcinoma (NPC) as amodel systemto test this hypothesis. Experimental Design: Plasma RNA integrity was determined using the ratio of the concentrations of transcript sequences corresponding to the 3V to those from the 5V end of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Transcript concentrations were measured using real-time quantitative reverse transcription-PCR assays targeting the 5V and 3V regions. We analyzed the plasma RNA integrity in 49 untreated NPC patients and 53 healthy controls.We also assessed the plasma samples from 19 NPC patients before and after radiotherapy to further show the clinical potential of this marker. Results: The 3V to 5V GAPDH ratio was significantly lower in the plasma of untreated NPC patients when compared with healthy controls (0.0252 versus 0.0485, P = 0.024). Statistical analysis showed that plasma GAPDH ratio was correlated with tumor stage but not with sex and age. Moreover, 14 of 19 NPC patients (74%) showed significant increase in the plasma GAPDH ratio following radiotherapy (P = 0.003). All of these patients were in clinical remission after treatment. Conclusions: Our findings suggest that NPC is associated with disturbances in the integrity of cell-free circulating RNA, raising the possibility that measurement of plasma RNA integrity may serve as a usefulmarker for the diagnosis andmonitoring of malignant diseases.
Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.
Wen Yang, Botoul Maqsodi, Yunqing Ma, Son Bui, Kimberly L. Crawford, Gary K. McMaster, Frank Witney, and Yuling Luo
Genospectra, Inc., Fremont, CA, USA
BioTechniques 40:481-486 (2006)
Formalin-fixed, paraffin-embedded (FFPE) tissues represent an important source of archival materials for gene expression profiling. We report here the development of a modified branch DNA assay that allows direct quantification of messenger RNA (mRNA) transcripts in homogenates from FFPE tissue sections without the need for RNA isolation and reverse transcription into cDNA. Formalin fixation essentially has no effect on the branch DNA assay, and RNA degradation only marginally reduces the signal by 2- to 3-fold. Under the same conditions, formalin fixation and RNA degradation greatly reduces real-time reverse transcription PCR (RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold, respectively. Although both technologies can generate biologically meaningful expression profiles from FFPE human lung tumor specimens, the branch DNA assay is more sensitive than real-time RT-PCR under the conditions tested. Our results therefore suggest that the branch DNA assay is an ideal tool for retrospective analysis of gene expression in archival tissues.
Quantification of mRNA in Whole Blood by Assessing Recovery of RNA and Efficiency of cDNA Synthesis.
Masato Mitsuhashi, Shigeru Tomozawa, Katsuya Endo, and Atsushi Shinagawa
Background: Current gene expression analysis relies on the assumption that the isolated RNA represents all species of mRNA in proportions equal to those in the original materials. No system is available for absolute quantification of mRNA. Methods: We applied whole blood to 96-well filterplates to trap leukocytes. Lysis buffer containing cocktails of specific reverse primers and known concentrations of synthetic external control RNA (RNA34) was added to filterplates, and cell lysates were transferred to oligo(dT)-immobilized microplates for hybridization. We then synthesized the cDNA in the oligo(dT)-immobilized microplates from these primer sites and used the cDNA for real-time PCR. RNA34 acted as a universal control, and gene amplification results were converted to quantities of mRNA per microliter of whole blood after the recovery of RNA34 in each sample was determined. Results: Under fully optimized conditions, both added RNA34 and native mRNA species exhibited 10% recovery from whole blood to real-time PCR. When whole blood was stimulated ex vivo, changes in gene expression as low as 30%–40% were detected with statistical significance, and the experimental CVs were low (10%–20%). Conclusion: This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression–based molecular diagnostics.
Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas.
Anne E. Mullin, Galina Soukatcheva, C. Bruce Verchere, and Janet K. Chantler
University of British Columbia, Vancouver and Child and Family Research Institute, Vancouver, BC, Canada
A technique to isolate high-quality intact RNA from murine pancreas is described. This technique involves in situ ductal perfusion of the pancreas with an RNase inhibitor prior to removal of the organ for RNA extraction. In this way, the pancreatic RNases are inhibited in situ allowing good yields of intact RNA, suitable for studies on pancreatic gene transcription by real-time PCR or microarray anaysis, to be obtained in a reliable way.
Noh Jin Park, Yang Li, Tianwei Yu, Brigitta M.N. Brinkman, David T. Wong
Background: We have previously shown that human mRNAs are present in saliva and can be used as biomarkers of oral cancer. In this study, we analyzed the integrity, sources, and stability of salivary RNA. Methods: We measured the integrity of salivary RNA with reverse transcription followed by PCR (RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites into the oral cavity, we used RT-PCR analysis of salivary RNA from the 3 major salivary glands, gingival crevice fluid, and desquamated oral epithelial cells. We measured stability of the salivar bneta-actin RNA by RT-qPCR of salivary RNA incubated at room temperature for different periods of time. We measured RNA association with other macromolecules by filtering saliva through pores of different sizes before performing RT-qPCR. To assess RNA–macromolecule interaction, we incubated saliva with Triton X-100 for different periods of time before performing RT-qPCR. Results: In most cases, we detected partial- to fulllength salivary mRNAs and smaller amounts of middle and 3 gene amplicons compared with the 5. RNA was present in all oral fluids examined. Endogenous salivary beta-actin mRNA degraded more slowly than exogenous -actin mRNA, with half-lives of 12.2 and 0.4 min, respectively (P <0.001). Salivary RNA could not pass through 0.22 or 0.45 m pores. Incubation of saliva with Triton X-100 accelerated degradation of salivary RNA. Conclusions: Saliva harbors both full-length and partially degraded forms of mRNA. RNA enters the oral cavity from different sources, and association with macromolecules may protect salivary RNA from degradation.
Del Aguila EM, Dutra MB, Silva JT, Paschoalin VM. Departamento de BioquÃmica, Instituto de QuÃmica, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, Bloco A, Lab. 545, CEP 21949-900, Rio de Janeiro (RJ), Brazil.
BMC Mol. Biol. 2005 Apr 156(1):9.
Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65 degrees C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step. Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.
John Ashkenas, James W. Dennis, and Chi Yip Ho
Samuel Lunenfeld Research Institute, Toronto, ON, Canada
BioTechniques 39:69-73 (July 2005)
Reverse transcription PCR (RT-PCR) is prone to false positives when contaminating DNA molecules are present at the start of a reaction. Contaminants that derive from earlier work using a given primer pair (carryover PCR products) are of particular concern when those primers are used routinely, as in clinical diagnostics or environmental monitoring. In addi-tion, contamination by genomic DNA can significantly interfere with quantitative and quali-tative analysis of RNAs by RT-PCR. Here we describe contaminant restriction (ConR), a method that can be used to neutralize carryover and genomic DNA contamination in RT-PCR studies. Restriction enzymes (REs) added to the amplification cocktail cleave contaminant DNA molecules while sparing the intended target nucleic acid. Restriction, reverse tran-scription, and amplification steps all take place in the same sealed vessel, thus avoiding any danger of recontamination. ConR eliminates carryover contamination in PCR without compromising target sequence amplification. Because the method is effective against both genomic and carryover contamination, it can be employed routinely in one-step RT-PCR, whatever the RNA target or the nature of the potential DNA contaminant. A variation of this decontamination method, amplicon primer site restriction (APSR), is effective specifically against carryover contamination. APSR, unlike ConR, can be applied during PCR-based amplification of DNA target molecules.
Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.
Katarina Cankar, Dejan Štebih, Tanja Dreo, Jana Žel and Kristina Gruden
Background: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislativerequirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methodsfor GMO detection are generally validated on certified reference materials that are in the form of powdered grainmaterial, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to foodprocessing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plantorigin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification.Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method mustensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNAextraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results: Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybeanmatrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assessvariability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliablequantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it waschosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extractiontechniques. The effect of PCR efficiency on the resulting GMO content is demonstrated.Conclusion: The crucial influence of extraction technique and sample matrix properties on the results of GMOquantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieveaccurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certainspecificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR.
Leslie Cler, Dawei Bu, Cheryl Lewis, David Euhus
Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9155, USA
Translational protocols in cancer and carcinogenesis often require isolation of genomic DNA from paucicellular clinical samples. DNA extraction methods for PCR-based applications should optimize the recovery of amplifiable DNA. We compared five methods for DNA extraction in paucicellular epithelial and lymphocyte samples using proportion of extractions producing amplifiable DNA and mean real-time PCR Ct values for GAPDH as the endpoint measures. The methods included solid-phase DNA adsorption (QIAamp), sequential protein and DNA precipitation (Puregene), magnetic bead adsorption (Dynabeads), phenol–chloroform extraction, and single-step proteinase K digestion. In general, the performance of the three commercial kits was superior to either phenol–chloroform extraction or single-step proteinase K digestion. However, QIAamp and Puregene produced amplifiable DNA more frequently than Dynabeads for starting cell numbers !50,000. GAPDH Ct values for QIAamp extractions showed the greatest dynamic range and the best linearity across the range of starting cell numbers, but QIAamp was not statistically significantly superior to Puregene. Of the three commercial kits, Puregene is the least expensive. QIAamp and Puregene DNA extraction methods are well-suited for the preparation of paucicellular clinical samples for PCR-based assays.