ImProm-II™ Reverse Transcription System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit can be used to reverse transcribe RNA templates starting with either total RNA, poly(A)+ mRNA or synthetic transcript RNA	Anneal reaction at 25°C, and incubate for 5 minutes. Extend at 42°C for up to one hour Inactive at 70°C for 15 minutes

Upstream tips
This kit can be used to reverse transcribe RNA templates starting with either total RNA, poly(A)+ mRNA or synthetic transcript RNA
Protocol tips
Anneal reaction at 25°C, and incubate for 5 minutes. Extend at 42°C for up to one hour Inactive at 70°C for 15 minutes

Publication protocol

Total RNA from kidney tissue was extracted using the TRIzol reagent (Invitrogen) followed by purification on RNeasy midi columns (Qiagen, Valencia, CA, USA) including DNAse treatment. cDNA was synthesized from 2 µg of total RNA using the ImProm-II cDNA synthesis kit (Promega, Madison, WI, USA). Reverse transcriptase-polymerase chain reaction (RT-PCR) reaction was performed using Platinum Blue PCR mix (Invitrogen) as per manufacturer’s instructions. The primers used for RT-PCR are based on published sequences with the following modifications [46]. Thermal cycling conditions were 95 °C for 5 min (hot start), followed by 35 cycles of 95 °C for 15 s denaturation, and 57 °C for 1 min annealing and extension. The PCR products were electrophoresed on 1.5% agarose gel and bands visualized using Biorad Imaging System. The intensity of the bands were quantitated by densitometric readings in Imagelab. The expression of each gene was normalized to 18S RNA and calculated relative to control animal.

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