Samples were then washed once in PBS and processed for RNA extraction using TRIzol reagent, and cDNA using Tetro cDNA kit (Bioline), as per manufacturer's instructions.
Tetro cdna kit
Manufactured by Meridian Bioscience
The Tetro cDNA Synthesis Kit is a tool for the reverse transcription of RNA into complementary DNA (cDNA). It provides a streamlined method for the conversion of RNA into cDNA, which can then be used in various downstream applications such as PCR, qPCR, or cloning.
Automatically generated - may contain errors
4 protocols using tetro cdna kit
1
RNA Extraction and cDNA Synthesis
2
Or22a Expression Analysis in Innisfail 2 Line
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One hundred antennae were dissected from snap-frozen adults from the Innisfail 2 isogenic line before RNA was isolated using β-mercaptoethanol, chloroform, and phenol extraction. cDNA was synthesized using a Tetro cDNA kit (Bioline) and amplified using GoTaq (Promega) to test for Or22a3 expression using Or22a-specific primers (Or22a-F2 5′-CTC CCA CCT TCG TGG TAA TGA A-3′; Or22a-R 5′-CAA AAA TGG TTC CCG AAA AG-3′). Diluted plasmid DNA was used as template for primer specificity tests. Or22a+b primers (Or22a+b-F 5′-GAG AGA TGC CTT CAT TTA CTT GG-3′; Or22a+b-R 5′-ACC CCA TGA GAA TGA CTT CG-3′) were used to confirm the quality of the Innisfail 2 antennal cDNA preparation. To sequence Or22 transcripts expressed in the Innisfail 2 isogenic line, ten heads were dissected from snap-frozen adults followed by RNA isolation and reverse transcription-PCR using the Or22a+b primers. The amplicon was ligated with pGEM-T (Promega) and 27 clones containing Or22 inserts were sequenced.
3
Real-time qPCR: Quantification and Normalization
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Real-time qPCR was performed as previously described82 with slight modifications. Briefly, first-strand cDNA was synthesized using Tetro cDNA Kit (Bioline) with 1 μg total RNA. The cDNA served as a template for the real-time qPCR. Relative quantification (RQ) was obtained using forward and reverse primers designed using the ‘Assay Design Center’ available at the Roche website. Primers were mixed with the cDNA and FastStart Universal Probe Master (Rox; Roche Diagnostics GmbH) and Universal ProbeLibrary Probe (Roche). A full list of primers and probes used for each gene can be found in Figure S2 . Sv-18S (GenBank accession no. KF828103 ) served to normalize the quantification. PCR included 10 min incubation at 94 °C, followed by 40 cycles of 94 °C for 10 sec and 60 °C for 30 sec, with green fluorescence measurement on each cycle at 60 °C. Reactions were performed in Rotor-Gene Q (Qiagen). Relative quantification was calculated by equilibrating to the level of Sv-18S per sample and the sample with the lowest value (2−∆∆CT). Statistical analysis of the resulting RQs was performed in Partek GS using ANOVA, followed by Mann-Whitney U-test with P < 0.05 considered as statistically significant.
4
RNA Extraction and qPCR Analysis
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RNA was extracted using New England Biolabs Monarch RNA extraction kit as per manufacturer's protocol, extracted RNA was stored in -80 0 C. RNA was subjected for cDNA synthesis using the Tetro cDNA kit (Bioline), oligo dt primers was used to enrich for mRNAs from total RNA. Quantitative realtime polymerase chain reaction (qPCR) was carried out using sensiFast Hi-Rox qPCR kit (Bioline) on ABI step one plus PCR cycler using the ΔΔCt method. Data were normalized against GAPDH. Primers are listed in supplementary table 2.
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