6.5 RT qPCR

6.5 RT qPCR kdorfman Thu, 04/21/2022 - 20:08

One-step RT-qPCR

One-step RT-qPCR kdorfman Tue, 04/26/2022 - 14:18

Luna Universal One-Step RT-qPCR

NEB E3005S, 200 rxns

  • Prepare RNA of interest using desired RNA extraction and purification methods. Determine concentration by OD260 absorbance.
  • Make dilutions of RNA to be used for the standard curve. These should be prepared fresh before each experiment and can be diluted in either water or TE.

Reaction Setup: For best results, we recommend running each RNA standard and sample in triplicate.

COMPONENT 20 µl REACTION FINAL CONC
Luna Universal One-Step Reaction Mix (2X) 10 µl 1X
Luna WarmStart® RT Enzyme Mix (20X) 1 µl 1X
Forward primer (10 µM) 0.8 µl 0.4 µM
Reverse primer (10 µM) 0.8 µl 0.4 µM
Template RNA variable < 1 µg (total RNA)
Nuclease-free Water to 20 µl

  1. Thaw Luna Universal One-Step Reaction Mix and other reaction components at room temperature, then place on ice. After thawing completely, briefly mix each component by inversion, pipetting or gentle vortexing.

  2. Determine the total volume for the appropriate number of reactions, plus 10% overage and prepare assay mix of all components except RNA template accordingly. Mix thoroughly but gently by pipetting or vortexing. Collect liquid to the bottom of the tube by brief centrifugation.

  3. Aliquot assay mix into qPCR tubes or plate. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles.

  4. Add RNA template to qPCR tubes or plate. Seal tubes with flat, optically transparent caps; seal plates with optically transparent film. Care should be taken to properly seal plate edges and corners to prevent artifacts caused by evaporation.

  5. Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).

  6. Program real-time instrument with indicated thermocycling protocol (see table below). Ensure a plate read is included at the end of the extension step.

CYCLE STEP TEMP TIME CYCLES
Reverse Transcription 55°C1 10 minutes 1
Initial Denaturation 95°C 1 minute 1
Denaturation
Extension		 95°C
60°C		 10 sec
30 sec2 (+ read)		 40-45
Melt Curve 60-95°C3 various 1

Notes

  1. A 55°C RT step temperature is optimal for Luna WarmStart Reverse Transcriptase. To ensure best performance and full WarmStart activation, avoid using a temperature of < 50°C. ↩︎

  2. For Applied Biosystems real-time instruments use a 60 second extension step. ↩︎

  3. Follow real-time instrument recommendations for melt curve step. ↩︎

RT, qPCR

RT, qPCR kdorfman Tue, 04/26/2022 - 14:16

Separate RT & qPCR

RT 10 groups @ 6 rxns 2 groups @ 8 rxns

Equipment

Hot blocks at

  • 50°C (2 dry baths - this is the long step and there are at least 4x as many tubes as groups),
  • 65°C
  • 70°C
    reagent uL/rxn uL/8 rxn (+ xtra) uL/class notes
    Oligo dT 50 µM 1 1 15 180
    10 mM dNTP mix 2 1 15 180
    RNAse-free water 11 + 40 60 720 aliquot once for both RT and qPCR
    5X 1st strand buffer 4 60 720 comes with Superscript
    0.1M DTT 1 15 125 1500 | comes with Superscript
    RNaseOUT 3 1 8 96 Dispensed from freezer box during lab
    Superscript 4 1 8 96 Dispensed from freezer box during lab

864 rxns @ 10uL

(24 x 3 rxns per group x 12 groups)

Reagent uL per rxn uL per 75 rxns
2x SYBR Green master mix5 5 uL 375 uL
water <5 uL 375 uL
forward primer 0.5 uL
reverse primer 0.5 uL
cDNA ~1 uL

  1. Fisher FERSO131 Oligo dT 100 µM (=0.5µg/µL) 60 µL @ ~$50. Dilute to 50µM!! (for emergencies: 50 µM oligo(dT) Fisher stockroom: C1101 0.5µg/µL x 40µL = 20µg) - Order T(18) from Invitrogen??? ↩︎

  2. 14 µL each + 84 µL RNase-free water OR Fisher FERR0192 ↩︎

  3. Invitrogen 10777019 RNAse out 5KU @ $143 ↩︎

  4. superscript III 200U/µL Invitrogen 18080044 10KU @ $259

    qPCR ↩︎

  5. Thermo Fisher 4385612 ↩︎