real time RT-PCR Protocol Comparison

BoVir® - real time RT-PCR with Lysis Buffer & Reagents versus Competitor real time RT-PCR

Ear Notch Sample Preparation with BoVir^®

• Ready-to-use proprietary lysis buffer
• Incubate sample with lysis-buffer: 200 µl; 60º C for 1 hour and 95º C for 10 minutes
• Pipette homogenate directly into the RT-PCR mix, single or 10 pooled samples
• Ready for real time RT-PCR

Ear Notch Sample Preparation with Competitor real time RT-PCR

Homogenization in PBS overnight

1a. Homogenize tissue samples in 10-20 volumes of TRI reagent or

1b. Homogenize cultured cells in 1 ml TRI reagent per 10 cm monolayer culture or per 5 x 106

2. Incubate the homogenate for 5 minutes at room temp

3. (Optional) Centrifuge at 12,000 x g for 10 minutes at 4º C and transfer the supernatent to a new tube

RNA Extraction

1. Add 100 µl of BCP to 1 ml of homogenate and mix well

2. Incubate the homogenate for 5 minutes at room temp

3. Centrifuge at 12,000 x g for 10 minutes at 4º C

4. Transfer 400 µl aqueous phase to a new 1.5 ml microcentrifuge tube

Final RNA Purification

1. Add 200 µl of 100% ethanol and mix immediately

2. Pass the sample through a filter cartridge

3. Wash the filter twice with 500 µl of wash solution

4. Elute RNA with 100 µl elution buffer

•Ready for real time RT-PCR