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qRT-PCR
Last updated date: Aug 2, 2022 Views: 567 Forks: 0
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qRT-PCR protocol
- RNA was isolated with the RNeasy mini kit (QIAGEN, Hilden, Ger- many) and cDNA was synthesized by reverse transcription (Invitrogen).
- 1 ug of RNA was used for preparing cDNA.
- Further for qRT-PCR, we used 10ng of cDNA in 384 well format (10 ul reaction volume per well) for quantification. SYBR green protocol was followed for the qRT-PCR.
Step | Temp | Time |
|
Initial denaturation | 94 °C | 2 min |
|
Denaturation | 94 °C | 15 sec |
x 55 cycles |
Annealing, extension, and read fluorescence | 60 °C | 1 min |
- Relative quantification method used to compare the RNA quantity among different groups.
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
- Kumar, A and Honjo, T(2022). qRT-PCR. Bio-protocol Preprint. bio-protocol.org/prep1838.
- Kumar, A., Chamoto, K., Chowdhury, P. S. and Honjo, T.(2020). Tumors attenuating the mitochondrial activity in T cells escape from PD-1 blockade therapy. eLife. DOI: 10.7554/eLife.52330
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