1Department of Laboratory Medicine & Pathobiology, University of Toronto, 2Division of Urology, Sunnybrook Health Sciences Centre, Toronto, Canada, 3Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, Canada, 4Biological Sciences, Sunnybrook Research Institute
This article is a part ofJoVE Clinical and Translational Medicine. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Gordanpour, A., Nam, R. K., Sugar, L., Bacopulos, S., Seth, A. MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR). J. Vis. Exp. (63), e3874, doi:10.3791/3874 (2012).
MicroRNAs (miRNAs) are single-stranded, 18–24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences.
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.
1. Prostate Sample Collection
2. Isolating Total RNA, Including miRNA, from Samples
Note: Here we have used TRIzol Reagent for extracting RNA, however other kits that isolate small RNA-containing total RNA can also be used.
3. Reverse Transcription of RNA
4. Generating a Standard Curve
Note: a new standard curve must be generated for each gene of interest.
5. Real-time PCR for Detection of miRNA
Note: Within a study, the same calibrating sample should be used to maintain consistency of results.
6. Analyzing Data
7. Representative Results
An example of qPCR analysis on prostate samples is shown in Figure 3. Results are depicted numerically, as well as graphically. The graphs showing the expression levels of the reference gene, U6, begin exponential amplification at about cycle 20, while expression of the target gene, miR-98, showed delayed amplification approximately at cycle 25. The data from this experiment was exported as text file and analyzed by RelQuant analysis software. Positions of the capillaries containing the calibrator and samples are specified. Figure 4 illustrates how the calibrator is set to be 1, and the expression of other samples relative to the calibrator.
Figure 1. Various steps in miScript reverse-transcription and Real-time PCR.
Figure 2. A standard curve is generated by using a series of dilutions of 2-fold, 10-fold, 50-fold, 250-fold, and 1250-fold the original cDNA sample.
Figure 3. Roche Molecular Biochemicals LightCycler Software shows the entire information of the experiment graphically and by text. Quantitative Real-time PCR amplification plots show increased in fluorescence from different samples.
Figure 4. Data were quantified using RelQuant LightCycler analysis software. Usually, three replicates of samples are analyzed as a group and samples that produce clearly inconsistent results are excluded and mean concentrations and standard deviations of the triplicate is calculated.
Aberrant expressions of some miRNAs have been consistently found in prostate tumors when compared to normal tissue10, and some of these miRNAs have been named as potential novel therapeutic agents against prostate cancer11. Hence the aberrant expression levels of miRNAs can be useful diagnostic and/or prognostic biomarkers. The Real-Time qPCR methodology presented here provides an assay for accurate quantification of miRNA levels in prostate tumor tissues. The miScript PCR system used can detect single nucleotide differences between mature miRNAs. The miScript miRNA qPCR Assays, however, are not intended for detection of stem loop precursor miRNAs, for which different miScript Precursor Assays are available.
The reliability of this technique depends on the quality of the input RNA, therefore concentration, integrity, and purity of RNA should be tested prior to Real-Time PCR. Moreover, ribonucleases are very stable and readily degrade RNA, thus extra caution should be taken in handling of the RNA. All reactions should be set up on ice to minimize RNA degradation. RNase inhibitors can also be added to the reaction prior to reverse transcription. Gloves should be frequently changed, while sterile and disposable plasticware must be used throughout the procedure.
If there is no PCR product or the amplification curve is detected late in Real-Time PCR, try increasing the number of PCR cycles, and make sure that the cycling program includes activation of HotStarTaq DNA polymerase for 15 minutes at 95 °C. Low amplification might also be due to inadequate starting cDNA template, therefore try to increase the amount of cDNA. Late amplification can also represent a false positive.
No conflicts of interest declared.
This work was funded by the Canadian Cancer Society Research Institute, grant no. 019038.
|miScript Reverse Transcription Kit||Qiagen||218061|
|miScript Primer Assays||Qiagen||Experiment specific|
|miScript SYBR Green PCR Kit||Qiagen||218073|
|LightCycler 3.5 Real-Time PCR System||Roche Group|
|Light Cycler Capillaries||Roche Group||04929292001|
|NanoDrop 1000 spectrophotometer||Thermo Fisher Scientific, Inc.||2538|
|Agilent 2100 Bioanalyzer||G2943CA|