Total RNA was isolated from retina, as previously described [3 (link)]. Following QC/QA analysis of RNA, reverse transcription of 10 ng RNA was performed to generate cDNA using SuperScript Enzyme Mix and VILO Reaction Mix (Life Technologies, Grand Island, NY) as a dNTP source. Levels of specific cDNA transcripts were assessed using TaqMan Gene Expression Master Mix (Applied Biosystems, Forest City, CA) and 1 µL of TaqMan probes specific for PEDF (Catalog #Mm00441270_m1), PEDF-R (Catalog #Hs00386101_m1), and glyceraldehyde 3-phosphate dehydrogenase or gapdh (Catalog #Mm99999915_g1) on a 7900HT Fast Real-time PCR system in triplicate (Applied Biosystems). The Δ-CT method was used to determine gene expression, using gapdh as the control gene. All samples were run in duplicate.
Superscript enzyme mix
Manufactured by Thermo Fisher Scientific
The SuperScript Enzyme Mix is a reverse transcription reagent manufactured by Thermo Fisher Scientific. It is designed for the conversion of RNA to cDNA, which is a fundamental step in various molecular biology applications, such as gene expression analysis and RT-PCR.
Automatically generated - may contain errors
Lab products found in correlation
Vilo reaction mix, by Thermo Fisher Scientific (4 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Qiaamp rna blood mini kit, by Qiagen (1 mentions)
4200 tapestation system, by Agilent Technologies (1 mentions)
Quantstudio 6 flex, by Thermo Fisher Scientific (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Taqman 2x universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
5 protocols using superscript enzyme mix
1
Retinal PEDF Gene Expression Analysis
2
Retinal PEDF Gene Expression Analysis
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Total RNA was isolated from retina, as previously described [3 (link)]. Following QC/QA analysis of RNA, reverse transcription of 10 ng RNA was performed to generate cDNA using SuperScript Enzyme Mix and VILO Reaction Mix (Life Technologies, Grand Island, NY) as a dNTP source. Levels of specific cDNA transcripts were assessed using TaqMan Gene Expression Master Mix (Applied Biosystems, Forest City, CA) and 1 µL of TaqMan probes specific for PEDF (Catalog #Mm00441270_m1), PEDF-R (Catalog #Hs00386101_m1), and glyceraldehyde 3-phosphate dehydrogenase or gapdh (Catalog #Mm99999915_g1) on a 7900HT Fast Real-time PCR system in triplicate (Applied Biosystems). The Δ-CT method was used to determine gene expression, using gapdh as the control gene. All samples were run in duplicate.
3
Single-cell TCR repertoire analysis
4
DNAH11 Transcript Variant Analysis
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Total RNA was extracted from 1–3 mL EDTA-anticoagulated whole blood using QIAGEN QIAamp RNA Blood Mini Kits according to manufacturer instructions. The extracted total RNA was reverse-transcribed to cDNA using Invitrogen Superscript Enzyme mix according manufacturer instructions. M13-tailed PCR primers flanking exon 65 (DNAH11-e64a-F1, 5′-TGTAAAACGACGGCCAGT AGAATGTCCACCGAAAATGC-3′; DNAH11-e64b-F2, 5′-TGTAAAACGACGGCCAGT TCCATTCCACTAACCGAAGG-3′; DNAH11-e66-R1/2, 5′-CAGGAAACAGCTATGACC TGCCAATTTTGTGTGAAGGA-3′) and primers flanking the termination codon (DNAH11-e82-F1, 5′-TGTAAAACGACGGCCAGTCCGTGGACAGACAAGAAACC-3′ and DNAH11-3′UTR-R1, 5′-CAGGAAACAGCTATGACCGACTTTCACTCTAAGGATAGCGTTG-3′) were used to PCR-amplify and Sanger-sequence the cDNA. cDNA product was visualized using the Agilent 4200 Tapestation system. Soft Genetics Mutation Surveyor (v5.1.2) was used to analyze Sanger sequence data, which was aligned against the DNAH11 NCBI Reference Sequence NM_001277115. Alamut Visual v2.11 was used to assess the transcript variants, including splicing effect prediction with SpliceSiteFinder-like, MaxEntScan, NNSPLICE, and GeneSplicer (Shapiro and Senapathy 1987 (link); Reese et al. 1997 (link); Pertea et al. 2001 (link); Yeo and Burge 2004 (link)).
5
NKT Gene Expression Analysis by qPCR
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Gene expression was measured in NKTs by quantitative polymerase chain reaction (qPCR) with specific primers and probes and normalized to the 18 S rRNA gene expression. Briefly, RNA was extracted from NKTs (RNeasy Plus Kit, Qiagen) and 1 µg of RNA was used for reverse transcription (5X VILO Reaction Mix and 10X SuperScript Enzyme Mix from Invitrogen). For the qPCR reaction (TaqMan 2X Universal PCR Master Mix from Life Technologies) 20 ng of cDNA were used in duplicates. The relative expression was calculated as follows: 2^- [(CTgene – CT18S) – CTVα24 in NT-NKTs]. Data acquisition was performed on a QuantStudio 6 Flex from Life Technologies using the QuantStudio Real-Time PCR software. The primers and probe for the 18S rRNA (Hs03003631_g1), CD62L (Hs00174151_m1), IL12β (Hs01011518_m1), IFNγ (Hs00989291_m1), FOXO1 (Hs00231106_m1) and TBX21 (T-bet, Hs00203436_m1) were purchased from Thermo Fisher Scientific.
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