RNA was extracted and quantified by reverse-transcription (RT) real-time quantitative (q) polymerase chain reaction (PCR) as previously described.21 (link),23 (link) In brief, RT was performed with 2 mg total RNA and AffinityScript reverse transcriptase (Agilent), and the cDNA quantified using SYBR Green qPCR reagent kit (Kapa Biosystems, London, UK) in a MX3000P light cycler (Agilent). Transcripts levels were calculated through comparison with calibrated standard curves and normalized to human ribosomal protein L28 mRNA. Primers used for RT-qPCR are listed in Online Supplementary Table S1. Epo protein was detected by ELISA according to the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA). Immunoblotting was performed as previously described21 (link) using the following primary antibodies: mouse monoclonal anti-HIF-1α (#610959; BD Transduction Laboratories, San Jose, CA, USA), rabbit monoclonal anti-HIF- 2α (#PAB12124; Abnova, Taipei, Taiwan), mouse monoclonal anti-HIF-b (D28F3; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-b-actin (A5441; Sigma Aldrich). Secondary antibodies were HRP-conjugated goat polyclonal anti-rabbit or anti-mouse IgG (#31460 and #31430, respectively; Thermo Fisher Scientific).
Sybr green qpcr reagent kit
Manufactured by Roche
Sourced in United Kingdom
The SYBR Green qPCR reagent kit is a laboratory product designed for quantitative real-time polymerase chain reaction (qPCR) analysis. The kit contains reagents necessary to perform quantitative gene expression studies.
Automatically generated - may contain errors
3 protocols using sybr green qpcr reagent kit
1
RT-qPCR and ELISA quantification of hypoxia markers
2
Quantitative RT-PCR Transcript Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted by the guanidinium thiocyanate-acid phenol-chloroform method as described earlier.55 (link) Complementary DNA (cDNA) was obtained by reverse transcription (RT) with AffinityScript transcriptase (Agilent, Santa Clara, CA, USA) and quantitated using the SYBR Green qPCR reagent kit (Kapa Biosystems, London, UK) and a MX3000P light cycler (Agilent) using standard curves. Transcript levels were normalized to the levels of human ribosomal protein L28 mRNA. Primer sequences are listed in Supplemental Table S2.
3
Quantitative Analysis of OTUB1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from tissues using the guanidine thiocyanate-acid phenol-chloroform method as previously described. 30 The RNA was converted to cDNA by reverse transcription of 2 µg of total RNA using AffinityScript transcriptase (Agilent, Santa Clara, CA, USA). The cDNA was analyzed using the SYBR Green qPCR reagent kit (Kapa Biosystems, London, UK) in a MX3000P light cycler (Agilent). Transcript levels were normalized to mouse ribosomal protein S12 mRNA levels. OTUB1 RNA-sequencing data from various human organs (Figure S1A) were extracted from a previous analysis. 31 OTUB1 RNA-sequencing data from murine and human lung cells/tissue were obtained from the LungMAP consortium (https://lungm ap.net/; 1U01HL122638; downloaded on 04/12/2020; Figure 3A) and Lung Gene Expression in Single-cell (LungGENS; https://resea rch.cchmc.org/pbge/lungg ens/defau lt.html; Figure 3B-M). 32-34
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!