RNA was extracted and quantitative real time (RT)-PCR was performed as previously described [28 (link)]. Briefly, total RNA was extracted from the cells using STAT-60 reagent (TEL-TEST, Inc., Friendswood, TX) following directions of the manufacturer. First-strand cDNA was synthesized using TaqMAN Universal PCR Master MIX (Applied Biosystems, Branchburg, New Jersey). Relative quantification PCR was performed using the ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) by the ΔΔCt method, as described previously [28 (link), 46 (link)]. Relative abundance of γ-globin-mRNA was determined as compared to α-globin mRNA or 18S as internal controls. γ-globin mRNA was analyzed in progenitors cultured from >60 different subjects. Relative mRNA abundance for LSD1, BCL11A, and KLF1 was similarly determined, using 18S rRNA as an internal control. TaqMan® Gene Expression primers were utilized from Life Technology (Grand Island, NY). Primer information is as follows: human γ-globin (Hs00361131_g1), human β-globin (Hs00758889_s1); human α-globin (Hs00361191_g1), 18S (Hs9999901_s1); LSD1 (Hs01002741 m1); BCL11A (Hs01093199_m1); EKLF1 (Hs00610592_M1) [28 (link), 31 (link)].
Stat 60 reagent
Manufactured by Tel-Test
Sourced in United States
The STAT-60 reagent is a laboratory product designed for use in various analytical procedures. It serves as a core component in specific testing applications, though its detailed function and intended use should not be extrapolated beyond the provided factual information.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression primer, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cfx thermocycler, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Experion, by Bio-Rad (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Cfx system, by Bio-Rad (1 mentions)
Iscript cdna kit, by Bio-Rad (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using stat 60 reagent
1
Quantitative RT-PCR Analysis of Globin and Regulator Genes
2
Quantitative gene expression analysis in mouse hippocampus
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Total RNA from hippocampus of adult male and female mice (n = 6/group) was isolated using STAT 60 reagent (Tel-Test, Friendswood, TX, USA) and RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. RNA concentration was determined by absorbance at 260 nm using a spectrophotometer (Nanodrop 2000, ThermoFisher Scientific, Madison, WI, USA). Total RNA was reverse transcribed into cDNA using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Real-time qPCR was performed on the CFX thermocycler (BioRad, Hercules, CA, USA) using the SsoAdvanced Universal SYBR Green Supermix (BioRad, Hercules, CA, USA) using standard protocols [17 (link),54 (link),55 (link),56 (link)]. Biological replicate samples were run in triplicate. Quantification of candidate gene expression levels was calculated based on the threshold cycle (Ct) for each well using the provided software and normalized to PPP2r2p as endogenous control. Relative changes in gene expression were normalized to the control male group. To extend the findings from our RNA-Seq analysis, we designed primers for genes in categories overrepresented in our Gene Ontology analysis. Primer sequences are listed in Table 2 .
3
Total RNA Extraction from PFC
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Total RNA from PFC of experiment 2 was isolated using STAT 60 Reagent (Tel-Test, Friendswood, TX, United States) and RNeasy mini kit (Qiagen, Valencia, CA, United States) according to the manufacturer’s protocol. RNA concentration was determined by absorbance at 260 nm and RNA quality was assessed by Experion automated electrophoresis (Bio-Rad, Hercules, CA, United States) and 28S:18S ratios. All RNA RQI values were >9.0, and 260/280 ratios were between 1.9 and 2.1.
4
Prefrontal Cortex RNA Isolation
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Homogenized and divided tissue was immediately pelleted and flash frozen on liquid nitrogen. Total RNA was isolated from PFC tissue using STAT60 Reagent (Tel-Test, Friendswood, TX) and miRNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. RNA was quantified by measuring absorbance at 260 nm using a NanoDrop, and RNA integrity was assessed by electrophoresis using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). All samples obtained an RNA Integrity Number (RIN) between 8.4 and 9.3.
5
RNA Extraction and Northern Blot Analysis
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Total RNA was isolated from cells using STAT60 reagent according to manufacturer’s instructions (Tel-Test Inc.). RNA was electrophoresed in 0.8% agarose gel with formaldehyde and transferred to membranes. Membranes were blocked, and probed with p32-HPV31 probe. After a series of washes with various stringency buffers, membranes were examined by autoradiography [63 (link)].
6
Quantifying Myelin-Related Gene Expression
7
Quantification of PVN, Pituitary, and Liver RNA
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Brains were removed, and sagittal sections were taken for PVN microdissection. PVN, pituitary, and liver were quickly removed and frozen in liquid nitrogen and subsequently stored at -80°C. PVN and pituitary RNA were extracted using Direct-zol RNA Microprep Kit (Cat. No. R2062; Zymo Research). RNA from the liver was extracted using STAT-60 reagent (Tel-Test, Friendswood, TX). Then, total RNA was reverse transcribed using SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). TaqMan gene expression assays for all messenger RNAs (mRNAs) were purchased from Life Technologies (Carlsbad, CA). Quantitative PCR was performed in duplicate using the 800 HT thermal cycler (Life Technologies, Foster City, CA). Relative mRNA levels were calculated using the standard curve method and normalized to the level of 18S ribosomal RNA
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