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#166028 RT-PCR - High Ct Values and Laser Capture

Posted by Quiksilver737 on 14 March 2014 - 09:13 AM in PCR, RT-PCR and Real-Time PCR

Hi,

 

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.

 

Brain Extraction:

 

1. Extract and flash freeze fresh rat brain in isopentane - store at -80C

 

Sectioning:

 

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)

 

RNA Extraction:

 

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

 

RT-PCR:

 

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). 

 

 

 

 





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