To analyze the effects of VIGS and overexpression on target genes expression, tissue samples from areas showing the silencing and enhancing phenotypes were collected. For controls, corresponding samples were collected from tissues infected by Agrobacterium carrying vectors with no host gene fragment insert, or from non-infected plants. Samples from three independent biological replicates were analyzed. Total RNA was extracted from crabapple leaves using the RNA plant plus Reagent (TIANGEN BIOTECH) according to the manufacturer’s instructions. DNase (TIANGEN BIOTECH) treatment was performed to remove any genomic DNA according to the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA using the Reverse Transcriptase M-MLV (RNase H−) kit (TaKaRa).
Reverse transcriptase m mlv rnase h kit
Manufactured by Takara Bio
Sourced in Japan, China
The Reverse Transcriptase M-MLV (RNase H-) kit is a laboratory tool used for the conversion of RNA into complementary DNA (cDNA). The kit contains the Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase enzyme, which lacks RNase H activity, enabling efficient and reliable cDNA synthesis from RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Trizol reagent, by Takara Bio (6 mentions)
Dnase 1, by Takara Bio (5 mentions)
Nanodrop one, by Thermo Fisher Scientific (4 mentions)
Rnaiso plus, by Takara Bio (3 mentions)
Sybr green pcr master mix, by Takara Bio (3 mentions)
Rnaplant plus reagent, by Tiangen Biotech (2 mentions)
Dnase, by Tiangen Biotech (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Lightcycle 96 real time pcr system, by Roche (2 mentions)
Sybr premix ex taq tli rnaseh plus, by Takara Bio (2 mentions)
Rnase free dnase 1, by Takara Bio (2 mentions)
Nanophotometer pearl, by Implen (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Cfx connect real time system, by Takara Bio (1 mentions)
Sybr green rt pcr kit, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Viia7 system software, by Thermo Fisher Scientific (1 mentions)
30 protocols using reverse transcriptase m mlv rnase h kit
1
Analyzing Gene Expression Changes
2
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from cells at 90% confluence by TRIzol (Invitrogen Life Technologies, Carlsbad, USA). Total RNA concentrations were measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA). According to the protocol, 2 μg of total RNA in each sample was reverse-transcribed by using the Reverse Transcriptase M-MLV (RNase H) Kit (Takara Biotechnology, Otsu, Japan). The cDNA after reverse transcription was diluted 5 times with ddH2O and stored at −20°C. qRT-PCR was performed using a SYBR Green RT-PCR Kit (Takara Biotechnology, Otsu, Japan), and specific primers were run in the CFX Connect™ Real-Time System. The relative expression of target genes was normalized to those of GAPDH and expressed as 2−ΔΔCt. The sequences of primers for qRT-PCR are listed in Table 1 .
3
Quantitative RT-PCR for GCAT1 Expression
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Total RNA from GCs and the cell lines was isolated using TRIzol reagent (Invitrogen) according to standard protocols. Approximately 700 ng of RNA were reverse transcribed using the PrimeScript RT reagent kit (Takara, Kyoto, Japan). For GCAT1 detection, reverse transcription was carried out using the reverse transcriptase M-MLV (RNase H−) kit (Takara) and specific reverse transcription primers. qRT-PCR labeled by SYBR green master mix (Takara) was performed on a LightCycler 480 system (Roche, USA). The primers for qRT-PCR are listed in Table S1 .
4
Silkworm Epidermis Transcriptome Analysis
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Epidermis samples were collected from every three silkworms after 6 hours of molting in the 4th instar, and total RNA was prepared using RNAiso Plus and reverse transcribed using the Reverse Transcriptase M-MLV (RNase H-) kit (TaKaRa, China) after treatment with DNase. The cDNA was diluted to 20 ng/μL and used as the template for qRT-PCR. The 20-μL reaction included 1 μL primer (10 μmol/L, S1 Table ), 1 μL cDNA, 10 μL 2×SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, China) and 8 μL ddH2O. After a rapid centrifugation step, quantitative reverse transcription PCR (qRT-PCR) was performed using a LightCycle 96 real time PCR system (Roche, Switzerland) with the reaction program below: a three-step reaction protocol of 45 cycles of 95°C for 10 s, 58°C for 10 s and 72°C for 10 s after a 10-min step of pre-degeneration, followed by melting. Relative expression was calculated using the 2–ΔΔCt method [25 (link)] with glyceraldehyde-3-phosphate dehydrogenase (GAPDH, BGIBMGA007490) as the reference gene. Melting analysis was performed to guarantee the qRT-PCR quality. The relative expression of 932VR and q-lp were compared, and RNA-Seq and qRT-PCR data were compared to analyze several key pathways.
5
Efficient RNA and DNA Extraction
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Total RNA was extracted using Trizol Reagent (Invitrogen, CA, USA) according to the manufacturer's protocol, treated with RNase-free DNase I (TaKaRa, Dalian, China) to degrade genomic DNA, and then frozen at −80°C. cDNA synthesis was performed with 1 μg total RNA and random hexamer primers using Reverse Transcriptase M-MLV (RNase H−) Kit (TaKaRa, Dalian, China) following the manufacturer's instructions. Genomic DNA was isolated from the muscle tissue using the traditional phenol/chloroform extraction method.
The quality and quantity of the RNA and DNA were evaluated by 1.5% agarose gel electrophoresis and spectrophotometry using NanoPhotometer Pearl (Implen, Munich, Germany).
The quality and quantity of the RNA and DNA were evaluated by 1.5% agarose gel electrophoresis and spectrophotometry using NanoPhotometer Pearl (Implen, Munich, Germany).
6
RNA and DNA Extraction from Muscle
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Total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The extracted total RNA was treated with RNase-free DNase I (TaKaRa, Dalian, China) to degrade genomic DNA and then frozen at −80°. cDNA was synthesized using 1 µg of total RNA and random hexamer primers with reverse transcriptase M-MLV (RNase H−) kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Genomic DNA was isolated from muscle tissues by the traditional phenol/chloroform extraction method. The quality and the quantity of RNA and DNA were evaluated by 1.5% agarose gel electrophoresis and spectrophotometry using NanoPhotometer Pearl (Implen, Munich, Germany).
7
Quantitative Gene Expression Analysis
8
Total RNA Extraction and Reverse Transcription
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Total RNA from leopard coral grouper was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and digested with DNase I (TaKaRa, Shiga, Japan) to remove potential DNA contamination. RNA concentration and purity were determined by NanoDrop One (Thermo, Waltham, MA, USA), and RNA integrity was verified by agarose gel electrophoresis. Only RNA samples with clear bands corresponding to 18S and 28S rRNA on the gel, an OD260/OD280 ratio between 1.8 and 2.0, and an OD260/OD230 ratio higher than 2.0 were used for subsequent experiments, and then frozen at −80 °C. The first-strand cDNA was synthesized by using 1 μg total RNA, random primers and Reverse Transcriptase M-MLV (RNase H-) Kit (TaKaRa, Shiga, Japan) [35 (link)].
9
Quantitative RT-PCR Analysis of nm2 Mutant C603 Larvae
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The heads of nm2 mutant and wildtype C603 larvae in the pre-molting stage were collected. Total RNA was extracted using RNAiso Plus (TaKaRa, China), according to the instructions of the manufacturer. After treatment with DNase I (TaKaRa, China), cDNA was synthesized using Reverse Transcriptase M-MLV (RNase H-) kit (TaKaRa, China), according to the instructions of the manufacturer, and was then diluted to 50 ng/μL to serve as the template for qRT-PCR. The 20-μL reaction system included 1 μL of primers (10 μmol/L, S1 Table ), 1 μL of cDNA, 10 μL of 2×SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, China) and 8 μL of ddH2O. After transient centrifugation, qRT-PCR was performed using a LightCycle 96 Real-time PCR System (Roche, Switzerland) with the following reaction program: three-step reaction protocol consisting of 45 cycles of 95°C for 10 s, 58°C for 10 s and 72°C for 10 s, after a 10-min step of pre-degeneration, followed by melting. The quality of the qRT-PCR product was tested through melting curve analysis, and relative expression was calculated using the 2–ΔΔCt method [19 (link)], with the average of three house-keeping genes, RPL3 (BGIBMGA013567), GAPDH (BGIBMGA007490) and BmActin3 (BGIBMGA005576), serving as the reference. The qRT-PCR results were compared with the DGE results to verify the accuracy of the DGE data.
10
Effector gene cloning in PVX vector
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The total RNA was extracted from mycelium of B. dothidea isolate ZY7 while using an RNA isolation kit (Omega Bio-Tek, Norcross, GA, USA). First-strand cDNA was synthesized from the extracted RNA using a Reverse Transcriptase M-MLV (RNaseH-) kit (Takara, Dalian, China). The open reading frame (ORF) sequences of candidate effectors (without SP), which were amplified from the cDNA of ZY7, and GFP were respectively inserted into the Potato Virus X (PVX) vector pGR107 [53 (link)] with a 3× flag-tag fused at the N-terminus by the method of digestion and connection using a ClonExpress II One Step Cloning kit (Vazyme Biotech, Nanjing, China). Table S3 lists the primers used for vector construction. After verification through sequencing, the generated construct was transformed into A. tumefaciens strain GV3101 by electroporation.
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