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REAL TIME PCR ANALYSIS

One of the most reliable methods for the determination relative of gene expression ratios is Real-Time QRT-PCR. Our unit is offering Real Time QRT-PCR.

Absolute quantification mostly done for the determination of viral copy numbers is also performed on Real-Time QRT-PCR platform.

a. Integrity of RNA Samples

The amount, integrity and purity of RNA samples are crucial for the quality, reliability and robustness of Real Time QRT-PCR. That is why our unit will rely on Agilent Bioanalyzer chromatogram or denaturing agarose gel profile for the evaluation of RNA samples. A260/A280  ratio of a good quality RNA sample should be between 1.9-2.1.

RNA samples should be brought to our unit on dry ice. If needed quality and quantity evaluation of RNA samples on Bioanalyzer can be performed in our unit.

It is possible to perform 50 analysis with 100 ul of cDNA synthesized from 1-2 ug of RNA sample. The RNA sample amount that should be submitted to our unit can be calculated based on this information.

Our unit can be consulted on RNA isolation methods. RNA isolation method should include a DNase treatment step.

b. Gene specific and Housekeeping genes primers

Primers which will be used in Real-Time qRT-PCR analysis:

  • Should be HPLC purified (HPSF purification of MWG or OPC purification of Metabion are also suitable)
  • Should be submitted at 10 pmol/ml concentration.
  • 10 pmol/ml of each primer is used for each reaction. The amount of primer that should be submitted to our unit can be calculated based on this information.
  • A selection of human housekeeping gene primer sets (ACTB, B2M, GAPDH, HMBS, HPRT1, RPL13A, SDHA, UBC, YWHAZ) are available in our unit.

 

Primer design tips:

1.Primers used in RT-PCR applications should be designed from different exons to ensure the elimination of amplification from possible DNA contamination.

  1. Primers Tm should be between 58-60oC.
  2. Primers %GC content should be between %30-80.
  3. The size of PCR amplicon should be between 100-150 bp.

Our unit can be consulted about primer design.

qRT-PCR applications are carried out using SYBR Green PCR kits. For studies that need the use of probes our unit should be informed and consulted.

c. Information that should be submitted along with the samples

  1. Bioanalyzer chromatogram or denaturing agarose gel profile and spectrophotometric readings of the RNA sample(s) should be attached to the request form.
  2. The sample information and analysis results will be processed with confidentiality.
  3. Samples which do not comply with the quality and quantity requirements will not be accepted for analysis.

d. Real-Time QRT-PCR results

 Real-Time QRT-PCR  results will be delivered to the customer via email or post (hard or digital copy on CD) in a  week depending on the sample number.