Bacterial samples were prepared similarly to RNA-seq preparation. A total of 10 genes (acsA, copZ, impA, piluA, pvdA, ntiB, moaB, phzR, fusA, and shiA) were selected for qRT-PCR analysis. The total RNAs was extracted from the cells of HT66 and HT66-FLUO using a total RNA isolation reagent (Invitrogen, Carlsbad, CA, United States), and reverse transcribed to cDNA using a TaKaRa RNA PCR Kit Ver.3.0. The resulting cDNA was amplified and quantified by RT-PCR with a Real Master Mix (SYBR Green) RT-PCR Kit (TaKaRa) on ABI Step-One Plus Real-Time PCR system. The rpoD gene was used as a reference. The expression level of mRNAs between HT66 and HT66-FLUO was compared by the 2-ΔΔCt method (Livak and Schmittgen, 2001 (link)).
Rna pcr kit ver 3
Manufactured by Takara Bio
Sourced in Japan
The RNA PCR Kit Ver.3.0 is a laboratory tool designed for the amplification and detection of RNA sequences. It provides a standardized protocol and necessary reagents to perform reverse transcription and subsequent polymerase chain reaction (RT-PCR) on RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Kod plus polymerase, by Toyobo (2 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Takara Bio (1 mentions)
Total rna isolation reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000 spectrophotometry, by Thermo Fisher Scientific (1 mentions)
Premier 5, by Sangon (1 mentions)
5 protocols using rna pcr kit ver 3
1
Comparative Gene Expression Analysis
2
Semi-quantitative RT-PCR Protocol
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Expression levels of mRNAs were determined using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) kit supplied by Takara RNA PCR kit Ver 3.0 (Takara, Shiga, Japan) and RT-PCR machine (TP850, Takara). Briefly, total RNAs were extracted with TRIzol (Invitrogen, Carlsbad, CA). RNA quantity and purity were determined by NanoDrop 1000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA). cDNAs were synthesized with AMV reverse transcriptase and random 9-mers and used as template for PCR amplification. The primer sets for amplification were listed in Supplementary Table 3 of SI . DNAs were amplified under the following conditions: denaturation at 95 °C for 5 min followed by 26 cycles of denaturation at 95 °C for 30 sec, annealing at 60 °C for 30 sec, and extension at 72 °C for 1 min. Relative quantity of amplified DNAs was analyzed using software supplied from TP850.
3
Notch1 Gene Expression in Spinal Cord Tissue
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Total RNA in spinal cord tissue was extracted with Trizol and reverse transcription was conducted using the RNA PCR kit Ver.3.0 (TaKaRa, Dalian, Liaoning Province, China) according to the manufacturer's instructions. Primers were designed by Premier 5.0 (Sangon Biotech, Shanghai, China); Notch1 gene: forward primer 5′-GCA GCC ACA GAA CTT ACA AAT CCA G-3′, reverse primer 5′-TAA ATG CCT CTG GAA TGT GGG TGA T-3′ (689 bp); β-actin: forward primer 5′-GTG GGG CGC CCC AGG CAC CA-3′, reverse primer 5′-CTT CCT TAA TGT CAC GCA CGA TTT C-3′ (170 bp). Amplification conditions were as follows: denaturation at 94°C for 5 minutes, 29 cycles of 94°C for 30 seconds, 56°C for 40 seconds and 72°C for 50 seconds, followed by extension at 72°C for 10 minutes. Reactions were preserved at 4°C. DNA products were examined by 2% agarose gel electrophoresis. Target gene expression is presented as the ratio of optical density of the gene product versus the internal reference.
4
Medaka RNA Isolation and mRNA Synthesis
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Total RNAs were isolated using TRIzol (Life Technologies) and were converted to cDNA using the RNA-PCR kit ver.3 (Takara Bio, Japan) followed by PCR using KOD plus polymerase (Toyobo, Japan). For mRNA production, PCR amplified full-length cDNAs (medaka YAP, 70KDaFN1a,b, ARHGAP18) were cloned into pCS2+ and for in situ hybrization medaka sox3 cDNA was cloned into pBluescript II SK(−). pCS2+myr-ARHGAP18 was constructed by adding the myristoylation sequence using oligonucleotides to produce myristoylated ARHGAP18 mRNA. mRNAs were synthesized using SP6 mMESSAGE mMACHINE Kit (Ambion, USA). Primer sequences are shown in Supplementary Table 5 .
5
Medaka RNA Isolation and mRNA Synthesis
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