qRT-PCR core

Summary:


Quantitative PCR allows for highly accurate, and cost effective measurement of fluorescent-labeled nucleic acids.  This system can be used to quantify cDNA isolated from mRNA and miRNA levels from cells or tissues.  The instrument also allows quantitative discriminations of DNA for genotyping, haplotype and SNP analyses.

Important notes to all users:


1) All new users must schedule training with Dr. Charles Nichols (cnich1@lsuhsc.edu) or Imran Mungrue (imungr@lsuhsc.edu), and obtain approval prior to use of qPCR machines.

2) Only approved plates and sealing film may be used as follows:

  • 384-well plate Roche Catalog# 04 729 749 001
  • 96-well plate, Roche Catalog# 04 729 692 001

3) Alert Imran Mungrue (imungr@lsuhsc.edu) to change the blocks (96-well or 384-well).

4) Users must sign up prior to use, to reserve a machine.

5) Always close the program by clicking the red X in the top right corner of teh Roche Software, and log off from windows when a run is completed.

 

RNA reagents:


RNA extraction:                    Option #1) Qiagen RNeasy (Cat# 74104)

Option #2) Invitrogen Trizol (Cat# 15596)

 

Nucleic acid quantitation: 

a) UV-Spectrometery: Nanodrop
b) Qubit RNA QC:
c) Agilent Tape station: R6K (Cat# ), Agilent High sensitivity R6K (Cat# )

 

cDNA Synthesis:                 Invitrogen/ Applied Biosystems/ABI High Capacity cDNA Reverse Transcriptase Kit (Catalog# 4368813)

qRT-PCR SYBR mix:          Roche 2X fast master mix (Cat # 04 887 352 001-50ml or Catalog #04 707 516 001-5ml)

Primers:                                 Design using primer blast, and synthesis from Integrated DNA Technologies.

Instrumentation:


  • qPCR:                        Roche Lightcycler 480-II: 96/384-well

    The LightCycler 480-II is a leading machine in the mid throughput range of instrumentation, boasting a silver thermal block with rapid heating (4.80C/sec) and cooling (2.50C/sec) rates.  The machine’s Therma-base technology allows precise experimental homogeneity minimizing well variability throughout the block.  The LightCycler 480 has 5 excitation filters (440, 465, 498, 533 and 618nm), 6 emission filters (488, 510, 580, 610, 640 and long pass filter: 660nm), and an internal barcode scanner.  Fluorescent dyes compatible with the system include: Cyan 500, FAM/SYBR, VIC/HEX, LC Red 610, LC Red 640, Cy5, Yakima Yellow, Cal Orange, Tamara, Cy3, Alexa 546, Cal Red 610, ROX, Texas Red and Alexa 647.  The LightCycler 480-II system includes software for complex analyses, including absolute quantification using the standard curve method, crossing point determination using the 2nd derivative max method, the proprietary “E-method” for quantitative assessments, and a genotype module for melt curve and endpoint calls.

     

    Nucleic acid QC:      Option #1) Agilent 2200 Tape station (capillary gel based)

                                        Option #2) Nanodrop (Spectrophotometry based)