qPCR Quantify cDNA, PCR, and Reaction Cleanup
Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo
Abstract
Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.
Steps
Prepare qPCR Master Mix; 9 μL Per Sample
Prepare qPCR Master Mix; 9 μL Per Sample:
| A | B |
|---|---|
| qPCR 2× master mix | 5.0 μL |
| H2O | 3.6 μL |
| qPCR primer mix | 0.4 μL (10 μM each qPCR-grade D5x/D7x mix) |
Mix, dispense into a 384-well qPCR plate.
Add 1µL , seal, mix.
Run qPCR, note Cq values.
PCR Amplify cDNA
| A | B |
|---|---|
| Prepare PCR on ice; 50 μL total per sample: | PCR conditions (cycle # depending on library): |
| 2× PCR master mix: 25.0 μL | 98 °C for 30 s |
| H2O: 7.5 μL | 98 °C for 15 s → 68 °C for 30 s → 72 °C for 40 s (×6 cycles) |
| 20 μM forward primer (D50x): 2.5 μL | 98 °C for 15 s → 72 °C for 60 s (×[qPCR Cq minus 9] cycles) |
| 20 μM reverse primer (D70x): 2.5 μL | 72 °C 1 min |
| Sample cDNA: 12.5 μL | 4 °C hold |
| Total cycle # for final PCR: 3 cycles less than the qPCR Ct or Cq of the 1:10 diluted sample |
Dispense into 8-well strips, add 12.5µL + 2.5µL and mix.
Perform PCR as indicated above.
SPRI Cleanup Library
Add 90µL suspension (do not separate) per 50 μL PCR reaction, mix, incubate at Room temperature for 0h 10m 0s (pipette mix 2–3× during incubation).
Magnetically separate, wash beads 2× with 75% EtOH remove the supernatant → air-dry beads 0h 5m 0s.
Resuspend in 20µL, let it sit for 0h 5m 0s, magnetically separate for 0h 5m 0s.
Transfer 18µL to new tubes.
