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

Table 2 Protocol used for analysis of serum miR-122

(A) Preparation of the RT reaction master mix
Component	Master mix volume per 15-μL reaction^a
100 mM dNTPs (with dTTP)	0.15 μL
MultiScribe^TM 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

^aEach 15-μL RT reaction consists of 7 μL master mix, 3 μL of 5× RT primer, and 5 μL RNA sample

ISSN: 1471-230X