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Table 2 Protocol used for analysis of serum miR-122

From: Analysis of association between circulating miR-122 and histopathological features of nonalcoholic fatty liver disease in patients free of hepatocellular carcinoma

(A) Preparation of the RT reaction master mix
Component Master mix volume per 15-μL reactiona  
100 mM dNTPs (with dTTP) 0.15 μL  
MultiScribeTM Reverse Transcriptase, 50 U/μL 1.00 μL  
10× Reverse Transcription Buffer 1.50 μL  
Rnase Inhibitor, 20 U/μL 0.19 μL  
Nuclease-free water 4.16 μL  
Total volume 7.00 μL  
(B) Performance of reverse transcription
Use the following parameter values to program the thermal cycler:
Step Time Temperature
Hold 30 min 16 °C
Hold 30 min 42 °C
Hold 5 min 85 °C
Hold 4
(C) Preparation of the qPCR reaction mix
Pipet the following components into each tube:
Component Single reaction  
TaqMan Small RNA Assay (x20) 1.00 μL  
Product from RT reaction 1.33 μL  
TaqMan Universal PCR Master Mix II (x2) 10.00 μL  
Nuclease-free water 7.67 μL  
Total volume 20.00 μL  
(D) Setting up the experiment or plate documentation and running the plate
In real-time PCR system software, create an experiment or plate document on real-time PCR system using the following parameters:
•Run Mode: Standard   
•Sample Volume: 20 μL   
•Thermal Cycling Conditions:   
  Enzyme Activation PCR CYCLE (40 cycles)
Step HOLD Denature Anneal/extend
Temperature 95 °C 95 °C 60 °C
Time 10 min 15 s 60 s
  1. aEach 15-μL RT reaction consists of 7 μL master mix, 3 μL of 5× RT primer, and 5 μL RNA sample