Mirneasy serum plasma reverse transcription kit

Total RNA was isolated from 250 lL serum according to the manufacturer's instructions of the miRNeasy extraction kit (Qiagen, Valencia, CA) by adding QIAzol lysis reagent (1 mL) then it was left for 5 min at room temperature. This was followed by adding 200 lL of chloroform and vortexing for 15 sec, the samples were incubated for 3 min at room temperature followed by centrifugation at 14,000 3 g at 48C for 15 min. An equal volume of 100% ethanol was added after removing the upper watery phase. Six hundred microliters of the solution were pipetted in miRNeasy Mini spin column and centrifuged at 8000 3 g for 60 sec. Then 600 lL of buffer RWT was added, followed by another centrifugation at 8000 3 g for another 60 sec. Then, 500 lL of buffer RPE was added prior to centrifugation at 8000 3 g for 2 min. The columns were placed in new collection tubes and centrifuged at full speed for 2 min. Fifty microliters of RNase-free water was placed directly into the columns followed by centrifugation for 1 min at 8000 3 g for RNA elution. Twenty microliters of eluted miRNA was reverse transcribed by incubation for 1 H at 428C, 3 min at 938C, and then maintained at 48C using the miRNeasy serum/plasma Reverse Transcription Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions (12) .