Количественные реального времени полимеразной цепной реакции (КПЦР) представляет собой быстрый и чувствительный метод для исследования уровня экспрессии различных микроРНК (миРНК) молекул в опухолевых образцах. Используя этот метод выражения сотен различных молекул микроРНК может быть усилен, количественно и проанализированы с той же кДНК шаблона.
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.
Аномальные проявления некоторых микроРНК были последовательно найдены в опухоли простаты по сравнению с нормальной тканью 10, и некоторые из этих микроРНК были названы как потенциальные новые терапевтические средства против рака простаты 11. Таким образом, аномальным уров?…
The authors have nothing to disclose.
Эта работа финансируется Канадским онкологическое общество Научно-исследовательский институт, грант №. 019038.
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 |