Total RNA was extracted using TRIzol Reagent (Life Technologies, Carlsbad, CA) from the retinas of 3-week-old WT and GLAST+/− mice 24 hours after vehicle or GGA was administrated. Reverse transcription was performed with GoScrip Reverse Transcriptase (Promega, Madison, WI) and oligo dT(20) primers following the manufacturer’s instructions. The TaqMan probe for mouse HSP70 was purchased from Life Technologies. Real-time PCR was performed using the GoTaq qPCR Master Mix (Promega), THUNDERBIRD Probe qPCR Mix (TOYOBO, Tokyo, Japan) and StepOnePlus™ Real-Time PCR System (Life Technologies). β-actin (sense 5′-CAT CCG TAA AGA CCT CTA TGC CAA C-3′, anti-sense 5′-ATG GAG CCA CCG ATC CAC A-3′) was used as endogenous control. Threshold cycle (CT) was determined automatically and relative changes in mRNA expression were calculated using the ΔΔCT values (Livak and Schmittgen, 2001 (link)). All PCR reactions were repeated in triplicate and the average values were used in statistical analyses.
Goscrip reverse transcriptase
Manufactured by Promega
GoScrip Reverse Transcriptase is a laboratory enzyme used to convert RNA into complementary DNA (cDNA) molecules. It performs the reverse transcription process, which is a key step in various molecular biology techniques, such as gene expression analysis and cDNA library construction.
Automatically generated - may contain errors
Lab products found in correlation
Gotaq qpcr master mix, by Promega (3 mentions)
Superprep cell lysis rt kit for qpcr, by Toyobo (3 mentions)
Thunderbird probe qpcr mix, by Toyobo (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Paxgene blood rna isolation kit, by Qiagen (1 mentions)
4 protocols using goscrip reverse transcriptase
1
HSP70 Expression in Retinal Cells
2
Quantifying Galectin-1 Gene Expression
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Total RNA isolation and reverse transcription were performed from cells using SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO, Tokyo, Japan) and from tissues using TRIzol (Life Technologies) and GoScrip Reverse Transcriptase (Promega, Madison, WI) with oligo dT(20) primers, as previously described44 (link). The following primers for genes were used: human LGALS1 (galectin-1; forward 5′- CGC TAA GAG CTT CGT GCT GAA C-3′, reverse 5′-CAC ACC TCT GCA ACA CTT CCA G -3′), human ACTB (ß-actin; forward 5′-CTG GAA CGG TGA AGG TGA CA -3′, reverse 5′-AAG GGA CTT CCT GTA ACA ATG CA -3′), mouse Lgals1 (forward 5′-TCC CCG AAC TTT GAG ACA TTC -3′, reverse 5′-GTC TCA GGA ATC TCT TCG CTT C -3′, probe 5′-ACC AGA CCA CAG GCC ATG ATT GAA -3′), and mouse Gapdh (glyceraldehyde-3-phosphate dehydrogenase; forward 5′-AGG TCG GTG TGA ACG GAT TTG -3′, reverse 5′-TGT AGA CCA TGT AGT TGA GGT CA -3′).
3
Total RNA Isolation, Reverse Transcription, and qPCR
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Total RNA isolation and reverse transcription were performed from cells using SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO) and from tissues using PureLink RNA Mini Kit (Thermo Fisher Scientifc) and GoScrip Reverse Transcriptase (Promega), as previously described.4 , 5 , 15 All primers are listed in Table S1 . Real‐time qPCR was performed using the GoTaq qPCR Master Mix (Promega) and StepOne Plus Systems (Thermo Fisher Scientific). Gene expression levels were calculated using the 2−ddCt method.
4
Gene Expression Analysis Protocol
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Total RNA isolation and reverse transcription were performed from cells using SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO, Tokyo, Japan) and from peripheral whole bloods using PAXgene Blood RNA Isolation Kit (QIAGEN, Venlo, The Netherlands) and GoScrip Reverse Transcriptase (Promega, Madison, WI) following the manufacturers’ protocols. The following primers for genes were used: ATP6AP2 [(P)RR; forward 5′-AGG CAG TGT CAT TTC GTA CC-3′ , reverse 5′-GCC TTC CCT ACC ATA TAC ACT C-3′ ], TNFA (TNF-α; forward 5′-ACT TTG GAG TGA TCG GCC-3′ , reverse 5′-GCT TGA GGG TTT GCT ACA AC-3′ ), CFD (forward 5’-GAC ACC ATC GAC CAC GAC-3′ , reverse 5′-GTT GAC TAT GCC CCA GCC-3′), LRG1 (forward 5′-GTT GGA GAC CTT GCC ACC T-3′ , reverse 5′-GCT TGT TGC CGT TCA GGA-3′ ), ACTB (β-actin; forward 5′-CTG GAA CGG TGA AGG TGA CA-3′, reverse 5′-AAG GGA CTT CCT GTA ACA ATG CA-3′) , and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; forward 5′-AGG TCG GTG TGA ACG GAT TTG-3′, reverse 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′ ). TaqMan probes for REN (prorenin) were purchased from Thermo Fisher Scientific. Real-time qPCR was performed using the GoTaq qPCR Master mix (Promega), THUNDERBIRD Probe qPCR Mix (TOYOBO), and StepOne plus Systems (Thermo Fisher Scientific).
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