Quantitative Real-Time PCR

GTC does not prepare the plates for users for QPCR runs. We only provide guidance, distribution of 384-well plates and covers and operation of the equipment at the user-reserved time. 

Register with GTC and request authorized access to the QPCR scheduling system by emailing us your kerberos ID. 

You can schedule time (1-2 hours/run) on the ViiA7 or ABI7900HT Real Time PCR here (authorized users only):

ViiA7 scheduling

Please note that new experiments should use the ViiA7.

384-well QPCR Guidelines

A) TYPICAL APPLICATIONS FOR 384-WELL QUANTITATIVE PCR

  1. Quantification of gene expression (qRT-PCR)
    Relative vs. absolute
  2. Protein-DNA interactions (Q-ChIP)
    (relative enrichment of promoter DNA-transcription factor binding)
  3. Detection of target sequence copy number
    Transgene copy number, viral load
  4.  Allele-specific detection
    SNP genotyping
    Mutation detection/representation (ASO QPCR)

Allele detection by amplicon dissociation profile

B) PRIMER DESIGN GUIDELINES:

  • Span exon-exon junctions wherever possible for gene expression analysis
    (RNA/cDNA) - use Ensemble Genome Browser for gene structure and Primer3 for primer design
  • Amplicon size 50-100 bp for SYBR Green, 60-150 bp for Taqman
  • GC content:
    TaqMan Probes: 30-80%
  • SYBR Green: 40-60%

    • Avoid low-complexity stretches

  • Tm: SYBR Green ~60oC
    Taqman hybridization probe: 10oC above amplification primer pair

C) PIPETTING RECOMMENDATIONS FOR 384-WELL PLATES

Total reaction volume per 384-well plate (ABI Cat# 4309849) is 10 uL:

5 uL 2x SYBR Green Mix

x uL primer mix (final conc. 50-500 nM each)

y uL template (0.1-5 ng cDNA, determined as 1-3% of input total RNA)

z uL H20

(x+y+z = 5 uL)

Replicate (ideally triplicate) reactions for each experimental condition, prepare master mixes wherever possible. For larger numbers of reactions use automated or semiautomated pipettes such as:

  • Eppendorf Repeater Pipettor
  • Matrix Impact®/Impact2™ Electronic Pipettors

D) Recommended consumables from Applied Biosystems (plates and covers available from GTC):

Cat # 4367659, 2x Power SYBR GREEN PCR Master Mix, 5 mL

Cat # 4309849, 384-WELL CLEAR REACTION PLATE, CASE OF 50

Cat # 4311971, OPTICAL ADHESIVE COVERS, box of 100