293Kb cells (2.5x105 cells/well in 12-well plates) were transfected with 1 μg of plasmid using Fugene HD (Promega). Total RNA was extracted 24h after transection using RNeasy Miniprep kit (Qiagen) with on-column DNase I treatment. First-strand cDNA was then synthesized using oligo-dT and Superscript III Reverse transcriptase system (Life technologies). Control lacking reverse transcriptase was performed for each RNA sample to monitor DNA contamination. Quantitative real-time PCR reactions (qPCR) were performed in iQ-SYBR green supermix using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Standard curves were generated using specific PCR amplicons and the obtained copy numbers were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) housekeeping gene (S2 Table ). Where indicated, transfected cells were treated with actinomycin D (Sigma) during 4.5h before RNA extraction.
Superscript 3 reverse transcriptase system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Superscript III Reverse Transcriptase system is a reverse transcriptase enzyme used for the conversion of RNA to cDNA. It is designed for high-sensitivity and high-specificity RNA-to-cDNA conversion.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (4 mentions)
Rnaseout, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Sv total rna isolation system, by Promega (2 mentions)
Rnaeasy mini kit, by Qiagen (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Oligo dt primer, by Qiagen (2 mentions)
Nuclease free water, by Qiagen (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Roche (2 mentions)
Ab 7500 fast system, by Thermo Fisher Scientific (2 mentions)
Quantifast sybr green pcr kit, by Qiagen (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Biosystems viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Sybr green real time pcr master mixes, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
48 protocols using superscript 3 reverse transcriptase system
1
Gene Expression Quantification by qPCR
2
Total RNA Extraction and Reverse Transcription
3
Klf6 Gene Expression Analysis in MA-10 Leydig Cells
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Total RNA was isolated from MA-10 Leydig cells using the RNeasy Plus Extraction Kit (Qiagen). First-strand cDNAs were synthesized from a 5 µg aliquot of the various RNAs using the Superscript III Reverse Transcriptase System (Life Technologies). PCRs were performed using the following Klf6-specific primers: forward (XbaI cloning site underlined) 5′-GCT CTA GAT ATC TTC AGA GTG AGC CCT GCT AC-3′; reverse (KpnI cloning site underlined): 5′-GGG GTA CCA GCC CCA TAG TTG AGA AGA TTC C-3′. The primers for αTubulin (Tuba1a) were described previously (Moisan et al. 2008) (link). Semi-quantitative PCRs for Klf6 and Tuba1a were performed on a T gradient thermocycler (Biometra, Göttingen, Germany) using Vent DNA polymerase (New England Biolabs, Beverly, MA, USA) under the following conditions: 5 min at 95°C followed by 30 cycles of denaturation (45 s at 95°C), annealing (45 s at 54°C) and extension (45 s at 72°C) with a final extension step of 5 min at 72°C. PCR products were then analysed by agarose gel electrophoresis and ethidium bromide staining.
4
Isolating and Quantifying High-Quality RNA from Antennae
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Total RNA was isolated from antennae samples by SV Total RNA Isolation System (Promega, Madison, USA) following the manufacturer’s protocol. The integrity of the RNA was checked by using 1.2% agarose gel electrophoresis and quantified using a ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) at OD260 nm. The high concentration (>800 ng/μL) of the total RNA showed that the high quality of the RNA sample meet the standard of reverse transcriptase reaction. The first strand cDNA was synthesized using the SuperScriptTM III Reverse Transcriptase System (Invitrogen, Carlsbad, CA, USA).
5
Tissue-Specific Expression Analysis in Silkworm
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To analyze tissue-specific expression, 12 tissues (head, midgut, fat body, Malpighian tubule, silk gland, wing disc, hemocyte, epidermis, nerve, muscle, and male and female reproductive organs) were dissected from silkworms on day 3 of the 5th instar. The wing disc and epidermis of B. mori from day 1 of the 4th instar larva to day 1 of the pupae samples were prepared to detect the temporal expression patterns. RNA was extracted and purified by an SV Total RNA Isolation System Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. After integrity confirmation by agarose gel electrophoresis and concentration determination by NanoDropTM 2000 (Thermo Fisher, Wilmington, DE, USA), 2 μg RNA was used to synthesize the first-strand cDNA using the SuperScriptTM III Reverse Transcriptase System (Invitrogen, Carlsbad, CA, USA).
6
Env gp120 C2-V5 Region Sequencing
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Viral RNA was isolated from cryopreserved plasma samples and purified using the QIAamp Viral RNA Extraction Kit (Qiagen) followed by reverse transcription using the SuperScript III Reverse Transcriptase System (Invitrogen). The Env gp120 C2-V5 (approximately 799 bp, HXB2 [accession number: K03455] positions 6883–7681) was amplified by nested touchdown PCR (primers listed in Table S1 ). Amplified DNA was purified using the MinElute Gel Extraction Kit (Qiagen), and ligated into a pCR4-TOPO sequencing vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen). Chemically competent One Shot MAX Efficiency DH5α E. coli (Invitrogen) were transformed with the prepared plasmids, and cultured overnight at 37 °C. Eighteen colonies were selected for colony PCR (M13F and M13R primers, Table S1 ), and the resulting products were purified using ExoSAP-IT (Affymetrix) and sequenced to generate forward and reverse reads (Source BioScience). Contigs were assembled and controlled manually using Geneious v9.0.561 (link) (HXB2 gp120 positions 661–1455, accession number K03455). Sequences were multiple aligned using MUSCLE62 (link), and then manually edited in MEGA v6.0663 (link). Sequences were controlled for intra-patient clustering by maximum-likelihood phylogenetics.
7
Quantifying BTLA mRNA Expression in PBMCs
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Total RNA was isolated from PBMCs using the RNAeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The superscript III Reverse Transcriptase system (Invitrogen, Carlsbad, CA) was used to synthesize cDNA. Real-time analysis of BTLA and beta-actin mRNA was performed using SYBR Premix Ex TaqTM II (TAKARA, Dalian, China) on the ABI 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. The primers for BTLA were purchased from Qiagen. The primers used for beta-actin were described elsewhere55 . Data were normalized to the expression of beta-actin using the 2−ΔΔCT method as described previously56 (link).
8
Quantitative PCR for Gene Expression
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Total RNA was extracted with TRIzol (Invitrogen) following the manufacturer’s instructions. RNA concentrations were determined with a Nano instrument (NanoDrop Technologies, Wilmington, DE). The first-strand cDNA was synthesized for each RNA sample using the Superscript III Reverse Transcriptase system (Invitrogen). Real-time quantitative PCR was performed on the iCycler (Biorad, UK) using the Quanti Tect SYBR Green PCR kit (Applied Biosystems). The forward and reverse primers for β-actin were designed using the Primer Premier software (Premier Biosoft International). The sequences of the PCR primer pairs were as follows: β-actin forward, 5′-GGA TGC AGA AGG AGA TCA CTG-3′ and reverse, 5′-CGA TCC ACA CGG AGT ACT TG-3′; TLR4 forward, 5′-AGT TTC CTG CAA TGG ATC AAGG-3′ and reverse 5′-CTG CTT ATC TGA AGG TGT TGC AC-3′; IL-6 forward, 5′-AGT GAG GAA CAA GCC AGA GC-3′ and reverse, 5′-CAG GGG TGG TTA TTG CAT CT-3′; IL-8 forward, 5′-GAC ATA CTC CAA ACC TTT CCA CCC-3′ and reverse, 5′-CCA GAC AGA GCT CTC TTC CAT CAG-3′. The human β-actin gene was used as an endogenous control for sample normalization. For each sample, the relative abundance of target mRNA was calculated from the obtained CΔt values for both the target and endogenous reference β-actin genes using the 2-ΔΔCt cycle threshold method.
9
Characterizing Olfactory Transcripts in Insect Heads
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We decided to study heads, as they contain most of the organs involved in chemosensory function and feeding, as well as most of the olfactory-associated proteins [47 (link),48 (link)]. Female heads were dissected on ice using a sterile scalpel and pooled in a 1.5 mL microcentrifuge tube (N = 5 per pool). Total RNA was extracted using the RNEasy Plant Mini Kit (QIAGEN, Hilden, Germany) and eluted in 50 μL of RNAse-free water. The integrity of the RNA samples was assessed using a 1.1% gel by denaturing formaldehyde agarose gel electrophoresis, and the concentrations were estimated by spectrophotometry at 260 nm (Epoch Microplate Spectrophotometer, Biotek), resulting in the range of 4.26–8.17 ng/μL of total RNA for all samples. DNA traces were removed from the samples by DNase treatment using Turbo DNase (Ambion). Single-stranded cDNAs were synthesized using the SuperScript III Reverse Transcriptase System (Invitrogen). All procedures were conducted following the manufacturer’s instructions.
10
RNA Extraction and Reverse Transcription
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