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Preprint

Quantitative real-time polymerase chain reaction (qRT-PCR)

YH Yong-Hyun Han
GR Gwendalyn J. Randolph

Last updated date: Sep 4, 2023 Views: 109 Forks: 0

original An abbreviated version of this protocol was published in Science in Jul, 2021
Enterically derived high-density lipoprotein restrains liver injury through the portal vein
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mABCA1Sequence (5'->3')
Forward primerGAAGAGAGCATGTGGAGTTCTT
Reverse primerTACTTTACCAGGCCCAGTTTG
mApoa1Sequence (5'->3')
Forward primerCTCTGGGTTCAACCGTTAGTC
Reverse primerTCTCCTGTCTCACCCAATCT
mCxcl2Sequence (5'->3')
Forward primerGACAGAAGTCATAGCCACTCTC
Reverse primerGCCTTGCCTTTGTTCAGTATC
mTNFSequence (5'->3')
Forward primerAATGGCCTCCCTCTCATCAGTT 
Reverse primerCCACTTGGTGGTTTGCTACGA 
mIl1bSequence (5'->3')
Forward primerAGAGCCCATCCTCTGTGACTCA 
Reverse primerTGCTTGGGATCCACACTCTCCA 
m18S rRNASequence (5'->3')
Forward primerGTAACCCGTTGAACCCCATT 
Reverse primerCCATCCAATCGGTAGTAGCG 
mMcp1Sequence (5'->3')
Forward primerCACTCACCTGCTGCTACTCA 
Reverse primerGCTTGGTGACAAAAACTACAGC 
mIl6Sequence (5'->3')
Forward primerGAACAACGATGATGCACTTGC 
Reverse primerTCCAGGTAGCTATGGTACTCC 
mCol1a1Sequence (5'->3')
Forward primerGACATCCCTGAAGTCAGCTGC 
Reverse primerTCCCTTGGGTCCCTCGAC 
mSaa1Sequence (5'->3')
Forward primerGAGGACATGAGGACACCATTG
Reverse primerCAGTCCAGGAGGTCTGTAGTAA
mActa2Sequence (5'->3')
Forward primerCCATCATGCGTCTGGACTT
Reverse primerGGCAGTAGTCACGAAGGAATAG
mTjp1Sequence (5'->3')
Forward primerCTGATGGTGCTCTGCCTAAT
Reverse primerTAGTCGCAAACCCACACTATC
mOclnSequence (5'->3')
Forward primerGCTCTTTGGAGGAAGCCTAAA
Reverse primerGCTGCTCTTGGGTCTGTATATC

cDNA was synthesized using the high-capacity cDNA reverse transcription kit from 1 ug of RNA (Applied Biosystems, #4368814). qRT-PCR experiments were performed using ABI StepOnePlus Real-Time PCR machine with specific primers (Applied Biosystems). Primer sequences are available upon request. The relative transcriptional expression of target genes was evaluated by the Eq. 2−DCt (DCt = Ct of target gene minus Ct of 18S rRNA). Relative transcription,where plotted, was calculated with the mean of the control group set as 1.

Copyright: © 2023 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Han, Y and Randolph, G(2023). Quantitative real-time polymerase chain reaction (qRT-PCR). Bio-protocol Preprint. bio-protocol.org/prep2412.
  2. Han, Y., Onufer, E. J., Huang, L., Sprung, R. W., Davidson, W. S., Czepielewski, R. S., Wohltmann, M., Sorci-Thomas, M. G., Warner, B. W. and Randolph, G. J.(2021). Enterically derived high-density lipoprotein restrains liver injury through the portal vein. Science 373(6553). DOI: 10.1126/science.abe6729

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