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Maxima h minus cdna synthesis master mix

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Maxima H Minus cDNA Synthesis Master Mix is a ready-to-use reagent for the synthesis of first-strand cDNA from total RNA or poly(A)+ RNA. The master mix contains Maxima H Minus Reverse Transcriptase, RNase Inhibitor, and optimized buffer components.

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Lab products found in correlation

Powerup sybr green master mix, by Thermo Fisher Scientific (19 mentions) Trizol reagent, by Thermo Fisher Scientific (14 mentions) Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (13 mentions) Trizol, by Thermo Fisher Scientific (11 mentions) Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (10 mentions) Rneasy mini kit, by Qiagen (8 mentions) Rneasy plus mini kit, by Qiagen (5 mentions) Powerup sybr green reagent, by Thermo Fisher Scientific (5 mentions) Dsdnase, by Thermo Fisher Scientific (4 mentions) Nucleospin rna isolation kit, by Macherey-Nagel (4 mentions) Sybr green master mix, by Thermo Fisher Scientific (4 mentions) Rneasy micro kit, by Qiagen (3 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions) Ssoadvanced universal sybr green supermix, by Bio-Rad (3 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions) Viia7, by Thermo Fisher Scientific (3 mentions) Green taq mix, by Vazyme (2 mentions) Quantitect primer assay, by Qiagen (2 mentions) Lightcycler 96, by Roche (2 mentions)

70 protocols using maxima h minus cdna synthesis master mix

1

Liver RNA Extraction and qRT-PCR Analysis

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Total RNA was extracted from mouse liver tissue using TRIzol (Invitrogen, catalog no. 15596026) following manufacturer’s recommendation. Total RNA (1 μg) was reverse-transcribed to synthesize cDNA using Maxima H Minus cDNA Synthesis Master Mix (ThermoFisher Scientific, catalog no. M1662), according to manufacturer’s protocol. cDNA (1:10) was used for quantitative real-time PCR using PowerUp SYBR Master Mix (ThermoFisher Scientific, catalog no. A25741) and ran on a QuantStudio 3 Real-Time PCR System. Gene expression was normalized to 18S rRNA (1:500 cDNA). Primer sequences used for gene expression analyses were as follows: mDbp Fwd: 5’-AATGACCTTTGAACCTGATCCCGCT-3’, Rev: 5’- GCTCCAGTACTTCTCATCCTTCTGT-3’. mBmal1 Fwd: 5’-GCAGTGCCACTGACTACCAAGA-3’, Rev: 5’- TCCTGGACATTGCATTGCAT-3’. mPer1 Fwd: 5’-ACCAGCGTGTCATGATGACATA-3’, Rev: 5’- GTGCACAGCACCCAGTTCCC-3’. mCpt1a Fwd: 5’-TGACTGGTGGGAGGAATACA-3’, Rev: 5’- AGTATGGCGTGGATGGTGTT-3’. The 18S rRNA Fwd: 5’-CGCCGCTAGAGGTGAAATTC-3’, Rev: 5’-CGAACCTCCGACTTTCGTTCT-3’.
Cervantes M., Lewis R.G., Della-Fazia M.A., Borrelli E, & Sassone-Corsi P. (2022). Dopamine D2 receptor signaling in the brain modulates circadian liver metabolomic profiles. Proceedings of the National Academy of Sciences of the United States of America, 119(11), e2117113119.
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2

Quantitative Analysis of MAFB and KAT5 Expression

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Total RNA was isolated from MG63 and U2OS cells via a TRIzol reagent (Sigma) after indicated treatment in each experiment. The extracted RNA was subjected to Maxima H Minus cDNA Synthesis Master Mix (Thermo) to obtain cDNAs, and subsequent quantification by an Applied Biosystem SYBR kit (Thermo). The relative expression of MAFB and KAT5 were normalized to the internal control GAPDH. Primers were listed:
MAFB, sense, 5’-GACGCAGCTCATTCAGCAG-3’, antisense, 5’-CTCGCACTTGACCTTGTAGGC-3’;
KAT5, sense, 5’-AACAAACGTCTGGATGAATGGG-3’, antisense, 5’-AGGAAGTCCGTTCTTAGTGGG-3’;
GAPDH, sense, 5’- sense, 5’-ACAACTTTGGTATCGTGGAAGG3’, antisense, 5’-GCCATCACGCCACAGTTTC-3’.
Wang Z., Song Y., Zhang H., Yang Y., Zhang S, & Wang W. (2022). Local anesthetic levobupivacaine inhibits stemness of osteosarcoma cells by epigenetically repressing MAFB though reducing KAT5 expression. Aging (Albany NY), 14(6), 2793-2804.
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3

Quantifying Viral Transcripts and Genomes

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An equal number of cells were harvested by centrifugation. RNA and DNA were extracted using IBI Mini Total RNA Kit (IB47323) and Mini Genomic DNA Kit (IB47202), respectively. 250 ng of total RNA was treated with DNAse and converted to complementary DNA (cDNA) using Maxima™ H minus cDNA Synthesis Master Mix (ThermoFisher, M1682). DNA and cDNA were diluted and amplified using iTaq™ Universal SYBR® Green Supermix (BioRad; 172–5124). Viral transcripts and genomes were compared to cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts and DNA respectively. qPCR primers have been previously described (IE Forward 5’-CGACGTTCCTGCAGACTATG-3’ Reverse 5’- TCCTCGGTCACTTGTTCAAA-3’ and GAPDH Forward 5’-GAGCCAAAAGGGTCATC-3’ Reverse 5’-GTGGTCATGAGTCCTTC-3’) (Hwang et al., 2011 (link); Juckem et al., 2008 (link)). Data was compared to untreated AD169 infections using the ΔΔCT method (Livak and Schmittgen, 2001 (link)).
Gelbmann C.B, & Kalejta R.F. (2019). The Golgi sorting motifs of human cytomegalovirus UL138 are not required for latency maintenance. Virus research, 270, 197646.
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4

RNA Extraction, cDNA Synthesis, and qPCR Analysis

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Total RNA was purified using the Total RNA Miniprep Kit (#AP-MN-MS-RNA, Axygen, Union City, CA, USA). Total RNA was treated with DNase I (#M1682, Thermo Scientific, Carlsbad, CA, USA) to remove genomic DNA contamination. Then, reverse transcription was performed using the Maxima H Minus cDNA Synthesis Master Mix (#M1682, Thermo Scientific, Carlsbad, CA, USA) by following the manufacturer’s protocol. Semiquantitative PCR was performed using Green Taq Mix (#P131, Vazyme Biotech, Nanjing, China). Quantitative real-time PCR was performed using Universal SYBR Green Fast qPCR Mix (#RK21203, Abclonal, Wuhan, China). The primers involved in these experiments are listed in Table S2.
Yan L., Sun Y., Guo J, & Jia R. (2023). PD-L1 Exon 3 Is a Hidden Switch of Its Expression and Function in Oral Cancer Cells. International Journal of Molecular Sciences, 24(9), 8193.
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5

Quantitative RT-PCR for Gene Expression

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Total RNA was extracted from cells using TRIzol (Thermo Fisher Scientific) as described by the manufacturer’s instructions. cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix (Thermo Fisher Scientific) using 1 μg of total RNA according to the manufacturer’s instructions. The cDNA was then used as template in quantitative real-time PCR using a SYBR Green Master Mix (Applied Biosystems). Gene expression was normalized to 18S ribosomal RNA. Primer sequences used for gene expression analysis are listed in Supplemental Table 1.
Pannunzio N., Rangel V., Sterrenberg J., Garawi A., Mezcord V., Folkerts M., Caulderon S., Wang J., Soyfer E., Eng O., Valerin J., Tanjasiri S., Quintero-Rivera F., Masri S., Seldin M., Frock R, & Fleischman A. (2023). Increased AID Results in Mutations at the CRLF2 Locus Implicated in Latin American ALL Health Disparities. Research Square.
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6

Quantifying Gene Expression in Lymphatic Endothelial Cells

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Gene expression in cell lysates or LECs sorted from mesenteric lymph nodes (mLNs), pooled from 4 to 5 mice per experiment) was measured by qPCR. MLN single-cell suspensions were created by mechanical dissociation followed by enzymatic digestion with DNase I, dispase II, collagenase D, and collagenase IV (Roche Diagnostics, Indianapolis, IN), from which LECs were sorted (podoplanin+/CD31+/CD45-) directly into lysis buffer (Qiagen, Hilden, Germany). RNA isolation was performed using RNeasy Mini and Micro kits for cell lysates and mLN LECs, respectively (Qiagen). cDNA was generated using Maxima H Minus cDNA Synthesis Master Mix and dsDNase (Thermo Fisher Scientific) using 8 μL of isolated RNA. qPCR was performed using QuantiTect SYBR Green PCR master mix (Qiagen) and pre-validated primer assays (Qiagen) using 1 μg of cDNA/reaction on a ViiA 7 Real-time PCR system (Thermo Fisher) per recommended protocol. After normalizing to β-actin expression, mRNA expression was measured as fold change using the ΔΔCt method. Samples with a value > 30 for β-actin were excluded to ensure mRNA quality. Each analysis was performed in triplicate.
Rehal S., Kataru R.P., Hespe G.E., Baik J.E., Park H.J., Ly C., Shin J, & Mehrara B.J. (2020). Regulation of lymphatic function and injury by nitrosative stress in obese mice. Molecular Metabolism, 42, 101081.
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7

Quantitative RT-PCR Analysis of Inflammatory Genes

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cDNA synthesis was performed using Maxima H Minus cDNA Synthesis Master Mix (Thermo Fisher Scientific) and RT–qPCR was performed as previously described29 (link). The following TaqMan Gene Expression Assays (Applied Biosystems) were used: Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), Tnf (Mm00443258_m1), Il10 (Mm01288386_m1), Il12b (Mm01288989_m1), Suclg1 (Mm00451244_m1), Auh (Mm00479363_m1), Ppia (Mm02342430_g1) and Rpl27 (Mm01245874_g1).
He W., Henne A., Lauterbach M., Geißmar E., Nikolka F., Kho C., Heinz A., Dostert C., Grusdat M., Cordes T., Härm J., Goldmann O., Ewen A., Verschueren C., Blay-Cadanet J., Geffers R., Garritsen H., Kneiling M., Holm C.K., Metallo C.M., Medina E., Abdullah Z., Latz E., Brenner D, & Hiller K. (2022). Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages. Nature metabolism, 4(5), 524-533.
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8

RNA Extraction and qRT-PCR Analysis

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Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction, and complementary DNA (cDNA) was prepared by using Maxima™ H Minus cDNA Synthesis Master Mix (Thermo Scientific, Rockford, IL, USA). Real‐time qPCR (qRT‐PCR; ViiA7; Life Technologies, Carlsbad, CA, USA) was performed in duplicates using predesigned primer sets (Quantitect Primer Assays, Qiagen, Germantown, MD, USA). Relative mRNA expression was analysed normalised to housekeeping genes, β‐actin or GAPDH.
Baik J.E., Park H.J., Kataru R.P., Savetsky I.L., Ly C.L., Shin J., Encarnacion E.M., Cavali M.R., Klang M.G., Riedel E., Coriddi M., Dayan J.H, & Mehrara B.J. (2022). TGF‐β1 mediates pathologic changes of secondary lymphedema by promoting fibrosis and inflammation. Clinical and Translational Medicine, 12(6), e758.
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9

Quantitative RT-PCR Analysis of BAT mRNA

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BAT mRNA was prepared from whole BAT tissue using Trizol (Invitrogen, 15596018). 1μg of RNA was used to synthesize cDNA using the Maxima H Minus cDNA Synthesis Master Mix (Thermo Scientific, M1662) and with an extension time of 60 min. at 50°C. qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1725270) using the following parameters:

95°C- 10 min.

95°C- 30 sec.

60°C- 1 min.

40 cycles of steps 2 and 3

95°C- 15 sec.

Amplification was performed using a QuantStudio 3 Real-Time PCR System. Analysis was performed using the 2ΔΔCT method and data were normalized relative to 18SrRNA expression. Primer sequences can be found in Table S6.
Dyar K.A., Lutter D., Artati A., Ceglia N.J., Liu Y., Armenta D., Jastroch M., Schneider S., de Mateo S., Cervantes M., Abbondante S., Tognini P., Orozco-Solis R., Kinouchi K., Wang C., Swerdloff R., Nathif S., Masri S., Magistretti P., Orlando V., Borrelli E., Uhlenhaut N.H., Baldi P., Adamski J., Tschöp M.H., Eckel-Mahan K, & Sassone-Corsi P. (2018). Atlas of Circadian Metabolism Reveals System-wide Coordination and Communication between Clocks. Cell, 174(6), 1571-1585.e11.
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10

Quantitative Gene Expression Analysis

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Total RNA was extracted using RNeasy mini kit (Qiagen). Purified RNA was reverse-transcribed using Maxima H Minus cDNA Synthesis Master Mix (Thermo Fisher). Quantitative real-time qPCR was performed on the AB7300 Detection System (Applied Biosystems, Foster City, CA, USA) using gene-specific primers and Power SYBR Green PCR Master Mix (Applied Biosystems). Expression of each gene was normalized to GAPDH, and quantified using 2-delta (ct) method.
Shi X., Zheng Y., Jiang L., Zhou B., Yang W., Li L., Ding L., Huang M., Gery S., Lin D.C, & Koeffler H.P. (2020). EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma. Nucleic Acids Research, 48(20), 11434-11451.
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