First strand buffer

Manufactured by Promega

Sourced in France, Netherlands

First strand buffer is a solution used in the first step of complementary DNA (cDNA) synthesis. It provides the necessary components, including salts and buffers, to optimize the reaction conditions for the reverse transcriptase enzyme to convert RNA into the first strand of cDNA.

Automatically generated - may contain errors

Lab products found in correlation

M mlv reverse transcriptase, by Promega (5 mentions) Random primer, by Promega (2 mentions) Evagreen dye, by Biotium (2 mentions) Total rna extraction kit, by Promega (2 mentions) Dntp mix, by Promega (2 mentions) Rnable, by Eurobio Scientific (1 mentions) M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Rnasin rnase inhibitor, by Promega (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Rnaseout, by Thermo Fisher Scientific (1 mentions) Ssofast evagreen supermix, by Bio-Rad (1 mentions) Oligo dt, by Promega (1 mentions) Improm 2 reverse transcriptase, by Promega (1 mentions) M mlv reverse transcriptase rnase h, by Promega (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)

8 protocols using first strand buffer

Quantitative RT-PCR for Endothelial Markers

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Total RNA was extracted from GSC and hCMEC using RNable (Eurobio, France) and verified by electrophoresis on Agilent 2100 Bioanalyzer (Agilent Technologies, France). cDNA was synthesized with 200 units of M-MLV Reverse Transcriptase (Invitrogen, France) in 15 μL of 1x first strand buffer (Promega, France), 2 mmol/L deoxynucleotide triphosphates in the presence of 40 units RNase inhibitor RNasin (Promega), 0.5 μg random primers (Promega), and 1 μg total RNA. Semiquantitative PCR amplifications were done with the following primer sequences: CD31 forward 5′-TCCGGATCTATGACTCAGGG-3′ and reverse 5′-ACAGTTGACCCTCACGATCC-3′; VE-cadherin forward 5′-TCCTCTGCATCCTCACTATCACA-3′ and reverse 5′-GTAAGTGACCAACTGCTCGTGAA-3′; ALAS forward 5′-TGCAGTCCTCAGGGCAGTCT-3′ and reverse 5′-TGGCCCCAACTTCCATCAT-3′ as control. The PCR conditions were as follows: 5 minutes at 94°C for denaturation, followed by 30 seconds at 94°C, 1 min at 60°C, and 1 min 30 sec at 72°C for 35 cycles and 7 min at 72°C for final elongation. The RT-PCR products were electrophoretically analyzed in 1% agarose and visualized by ethidium bromide staining.

El Hallani S., Colin C., El Houfi Y., Idbaih A., Boisselier B., Marie Y., Ravassard P., Labussière M., Mokhtari K., Thomas J.L., Delattre J.Y., Eichmann A, & Sanson M. (2014). Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature. BioMed Research International, 2014, 827327.

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Optimized RNA Extraction and Quantification

Cited 3 times

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For RNA extraction, cells were harvested by centrifugation at 700 rpm, the cell pellets were resuspended in diethyl pyrocarbonate (DEPC) treated buffer A (20 mM Tris-HCl, pH 7.9; 1.5 mM MgCl₂; 10 mM KCl and 0.5 mM DTT; 0.2 mM PMSF), incubated on ice for 10 min, and centrifuged at 3500 rpm for 5 min. The supernatant containing the RNA was subjected to phenol-chloroform extraction followed by ethanol precipitation of RNA by incubating 1h at −80 °C (17 (link)). Reverse transcription (RT) reactions were performed in a total volume of 25 µL of cDNA containing 500 ng of RNA, 2.4 µM of oligo dT (Promega), 100 units of MMLV reverse transcriptase, 1X first strand buffer (Promega), 100 µM each of dATP, dGTP, dCTP and dTTP (Invitrogen), 1 mM DTT, and 20 units of RNaseOut (Invitrogen). The cDNA was diluted to 100 µL. PCR was performed in a 10 µL reaction volume containing 5 µL diluted cDNA and gene specific primer pairs (Table 1). For qPCR analyses, the cDNA was amplified using SsoFast EvaGreen supermix (Bio-Rad) using CFX96 real-time PCR detection system. The qPCR results were analyzed using the CFX manager software. The experiments were repeated at least thrice with three replicates each time (16 (link), 46 (link)–48 (link)).

Hussain I., Bhan A., Ansari K.I., Deb P., Bobzean S.A., Perrotti L.I, & Mandal S.S. (2015). Bisphenol-A induces expression of HOXC6, an estrogen-regulated homeobox-containing gene associated with breast cancer. Biochimica et biophysica acta, 1849(6), 697-708.

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Quantifying Plant miRNA Expression

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Total RNA was isolated from flower buds (Solanum lycopersicum UC82 cv) injected with either LDHs (ctrl) or dsRNA-LDHs (AGL6sil or IAA9sil). The stem–loop qRT-PCR primers were designed according to [26 ]. Stem–loop RT reactions were performed following the thermal conditions reported in [27 ]. Briefly, the reaction mixtures were incubated at 16 °C for 30 min, followed by 60 cycles at 30 °C for 30 s, 42 °C for 30 s, and 50 °C for 1 s. At the end, the RT enzyme was inactivated at 85 °C for 5 min. The RT reaction in a total volume of 20 µl, contained 2 µg of RNA samples, 1 µL of 1 µM stem-loop RT primer, 1 µL of 10 mM dNTP mix, 4 µL 5X First-Strand buffer, 1.3 µl of 25mM MgCl₂, and 1 µL Improm II Reverse transcriptase (Promega). The relative expression level of target miRNAs was calculated using 2^−ΔΔCt method [28 ] normalizing against U6 (XR_003244923.1) as the reference gene. The list of primers used is reported in Additional file 5.

Molesini B., Pennisi F., Vitulo N, & Pandolfini T. (2023). MicroRNAs associated with AGL6 and IAA9 function in tomato fruit set. BMC Research Notes, 16, 242.

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Tissue-Specific Gene Expression Profiling

Cited 1 time

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Specific gene expression was assayed by RT-qPCR for heart, liver, brain, lung, and testis RNA previously extracted from broiler tissue samples [34 (link)]. Total RNA (500 ng) was added to a mastermix consisting of 1x First Strand Buffer (Promega Corp.), 0.5 μM dNTPs, 1 μM CT₂₃V primer and 100 U MMLV Reverse Transcriptase (Promega Corp.). The mixtures were incubated at 50°C for 50 min and the reaction was terminated at 65°C for 5 min. The first strand cDNA was then diluted into a qPCR mixture (as described above) containing 1x EvaGreen dye (Biotium Inc., Fremont, CA). Cycling was as for exonuclease assays (above) but were followed by a high resolution melt curve from 65 to 90^oC at 0.1^oC steps. RT-qPCRs were run in triplicate with TATA-box binding protein (TBP) gene as the reference, and all ΔΔCt values were relative to TBP [39 (link)]. Fold change was calculated using the ΔΔCt method [47 (link)].

Parveen A., Jackson C.D., Dey S., Tarrant K., Anthony N, & Rhoads D.D. (2020). Identification and validation of quantitative trait loci for ascites syndrome in broiler chickens using whole genome resequencing. BMC Genetics, 21, 54.

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Sunitinib-Treated Clear-Cell RCC RNA Isolation

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Frozen tissue from a selection of patients was available for RNA isolation, including clear-cell RCC patients pretreated with sunitinib (n = 12) and untreated patients as control (n = 5). Briefly, frozen tissue was lysed with TRIzol (Invitrogen, Lucern, Switzerland) and RNA was extracted with NaAC 4.5 pH (3 M), acidic phenol 4.5 pH (AM9720, Ambion Inc, Austin, TX, USA) and chloroform (0.1:1:0.2). RNA concentrations were measured using the NanoDrop-2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). One μg of total RNA was incubated for 5 min at 70 °C, and cDNA synthesis was performed for 1.5 h at 42 °C with 400 U of M-MLV reverse transcriptase RNase H (Promega, Leiden, the Netherlands) in 20 µl of 1 × first strand buffer (Promega), and 1 mM dNTPs in the presence of 10 U RNase inhibitor rRNasin (Promega) and 0.5 µg random primers (Promega). The reverse transcriptase activity was inactivated by incubation at 95 °C for 5 min and following addition of 1xTE up to a final volume of 50 µL the cDNAs were stored at -20 °C.

Nowak-Sliwinska P., van Beijnum J.R., Griffioen C.J., Huinen Z.R., Sopesens N.G., Schulz R., Jenkins S.V., Dings R.P., Groenendijk F.H., Huijbers E.J., Thijssen V.L., Jonasch E., Vyth-Dreese F.A., Jordanova E.S., Bex A., Bernards R., de Gruijl T.D, & Griffioen A.W. (2022). Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy. Angiogenesis, 26(2), 279-293.

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Quantifying Gene Expression in Broiler Tissues

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Specific gene expression was assayed by RT-qPCR for heart, liver, brain, lung, and testis RNA previously extracted from broiler tissue samples [35] . Total RNA (500 ng) was added to a mastermix consisting of 1x First Strand Buffer (Promega Corp.), 0.5 µM dNTPs, 1 µM CT 23 V primer and 100 U MMLV Reverse Transcriptase (Promega Corp.). The mixtures were incubated at 50°C for 50 min and the reaction was terminated at 65°C for 5 min. The first strand cDNA was then diluted into a qPCR mixture (as described above) containing 1x EvaGreen dye (Biotium Inc., Fremont, CA). Cycling was as for exonuclease assays (above) but were followed by a high resolution melt curve from 65 to 90 o C at 0.1 o C steps. RT-qPCRs were run in triplicate with TATA-box binding protein (TBP) gene as the reference, and all ΔΔCt values were relative to TBP [40] . Fold change was calculated using the ΔΔCt method [48] .

Parveen A., Jackson C.D., Dey S., Tarrant K.J., Anthony N.B., & Rhoads D.D. (2020). Identification and validation of quantitative trait loci for ascites syndrome in broiler chickens using whole genome resequencing.

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RNA Extraction and cDNA Synthesis from Diverse Tissues

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Total RNA was extracted from different tissues and from Hp and He of four groups using Total RNA Extraction Kit (Promega, Shanghai, China) according to the manufacturer’s protocol. Total RNA quality was assessed by agarose gel electrophoresis and Nano Drop ND-1000. The complementary DNA (cDNA) was synthesized in 20 μL reaction system including 3 μg total RNA (treated with DNase I), 2 μL random primers (10 mM), 4 μL 5× First-strand Buffer, 1 μL dNTP mix (10 mM), and 1 μL M-MLV reverse transcriptase (200 U/μL) (Promega, Shanghai, China). According to our previous data [38 (link),40 (link),49 (link)], the synthesized cDNA was diluted by 10-fold and 100-fold and stored at −20 °C until use.

Fang Z., Sun Y., Zhang X., Wang G., Li Y., Wang Y, & Zhang Z. (2019). Responses of HSP70 Gene to Vibrio parahaemolyticus Infection and Thermal Stress and Its Transcriptional Regulation Analysis in Haliotis diversicolor. Molecules, 24(1), 162.

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Total RNA Extraction and cDNA Synthesis

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Total RNA was extracted from different tissues using Total RNA Extraction Kit (Promega, Shanghai, China) according to the manufacturer's protocol. Total RNA quality was assessed by agarose gel electrophoresis and Nanodrop 2000 (Thermo Scientific). The complementary DNA (cDNA) was synthesized in a 20 μL reaction system including 1 μg total RNA (previously treated with DNase I), 2 μL random primers (10 mM), 4 μL 5× First-strand Buffer, 1 μL dNTP mix (10 mM), and 1 μL M-MLV reverse transcriptase (200 U/μL) (Promega, Shanghai, China). The synthesized cDNA was diluted and stored at −20°C until use.

Liao J., Zhang Z., Jia X., Zou Z., Liang K, & Wang Y. (2020). Transcriptional Regulation of Vih by Oct4 and Sox9 in Scylla paramamosain. Frontiers in Endocrinology, 11, 650.

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