MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

Summary

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Abstract

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.

Protocol

1. Prostate Sample Collection

1. Collect the prostate samples at the time of prostatectomy. The specimen is oriented using anatomic landmarks. The prostate and seminal vesicles are painted as follows: right side green, left side blue.
2. A random transverse midsection of the prostate is taken perpendicular to the rectal surface, frozen in liquid nitrogen, and stored at -80 °C⁹.
3. Banked slices of specimens are photocopied, oriented (anterior, posterior, right and left), q.......

Discussion

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 sin.......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number
TRIzol Reagent	Invitrogen	15596
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
Light Cycler Capillaries	Roche	04929292001
NanoDrop 1000 spectrophotometer	Thermo Scientific	2538
Agilent 2100 Bioanalyzer	G2943CA