My RT-PCR runs have been producing high Ct values (GAPDH: ~28-29, Cyclophilin: ~31, ARGP: ~33, NPY: ~33) and I need some help determining the cause.
Here is a short summary of the protocols I use.
1. Extract and flash freeze fresh rat brain in isopentane - store at -80C
1. Let brain acclimate to cryostat temp (-20C)
2. Wipe stage with RNase Zap
3. Set brain in Tissue Tek O.C.T compound - freeze at -20C for 40min
4. Section onto PEN membrane slides
5. Store at -80C
Laser Capture (Leica LMD 7000)
1. Wash tissue in RNase-free PBS for 5 min
2. Dehydrate in RNase-free 70% EtOH for 10min
3. Air dry at room temp for 30min
4. Cut tissue and collect into 0.5ml RNase-free tube
5. Add 50ul of RLT buffer (provided in Qiagen's RNeasy Mini Kit) to tube and store on dry ice until sample can be stored at -80C (~1hr)
1. For the extraction, I use Qiagen's RNeasy Mini Kit. I do add BME to the RLT buffer, and homogenize the cells by passing the sample through a 25 gauge needle 20-30 times. I wipe all surfaces down with RNase Zap, and try to stay as RNase-free as possible. All pipet tips and collection tubes are RNase-free.
2. The samples are stored at -80C
cDNA Reverse Transcription:
1. I use Applied Biosystems High-Capacity cDNA Reverse Transcription Kit.
2. Each sample contains:
4uL RT Buffer, 1.6uL dNTP, 8.4uL RNase-free H2O, 2uL reverse transcriptase and 20uL RNA sample
3. cDNA is stored at -25C
1. I use Life Technologies' Assays on Demand. The ratio for one well is:
1.25uL AOD mix
1.25uL nuclease-free H2O
12.5uL TaqMan Fast Advanced Master Mix
2. cDNA is diluted in nuclease-free water
Thank you for any information. I have a feeling that something is going wrong during the extraction process, or possibly during laser capture. A previous technician was able to get lower Ct values (~24-26 for the genes listed above) using the same protocols, although he was using a different LMD and RT-PCR machine (used same threshold values).