RT-PCR - High Ct Values and Laser Capture - (Mar/14/2014 )
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).
Your cDNA reaction seems a likely issue. The volumes do not add up to 40 ul, despite having 4 ul of RT buffer. Also, more than half of your volume is RNA, which is an invitation for reaction inhibitors. If you have even the slightest hint of ethanol in your RNeasy prep final elution, you will be adding lots of it to your RT reaction. I would run this reaction with half as much RNA, and I would be very sure I did the long, high speed, dry spin in a new tube suggested in the RNeasy manual.