According to the growth curve of the S. diastatochromogenes 1628, the wild-type and ppGpp0 strains were cultured to the early, mid-, or late exponential phases in the GYM medium at 28°C. The samples were harvested by centrifugation at 4°C and 4000 × g for 10 min. The total RNA was extracted by the Takara MiniBEST Universal RNA Extraction Kit (Dalian, TaKaRa, Japan), and reverse transcribed using the PrimeScript RT reagent kit (TaKaRa, Kusatsu, Japan). qPCR was conducted in a 20 μL volume with 100 ng cDNA as the template using the SYBR Premix Ex Taq GC kit (TaKaRa, Japan), and the thermocycling conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 30 s (primers are in Table S1 in Supplemental File 1). The 16S rRNA gene was used as the reference gene. Reverse transcription-quantitative (qRT)-PCR results were conducted in the Step One Plus Real-Time PCR system (Applied Biosystem), and the data were analyzed by the software Step One. The 16S rRNA gene processing protein (rimM) was used as a reference gene for normalization. The standard deviation (SD) indicates the standard deviation from three independent experiment replicates.
Sybr premix ex taq gc kit
Manufactured by Takara Bio
Sourced in Japan, United States
The SYBR Premix Ex Taq GC kit is a real-time PCR reagent designed for high-performance gene expression analysis. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, and a proprietary Taq polymerase, enabling efficient and sensitive detection of target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Abi 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Dig rna labeling kit, by Roche (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Applied biosystems 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Abi 7500 thermocycler, by Thermo Fisher Scientific (1 mentions)
Rnaprep pure cell bacteria kit, by Tiangen Biotech (1 mentions)
7500 sequence detection system, by Takara Bio (1 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Ariamx 3000p real time pcr system, by Agilent Technologies (1 mentions)
Sybr premix ex taq gc, by Takara Bio (1 mentions)
15 protocols using sybr premix ex taq gc kit
1
Transcriptional Analysis of S. diastatochromogenes 1628
2
Transcriptional Analysis of Saccharopolyspora erythraea
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Saccharopolyspora erythraea was grown for 2 days at 30 °C in seed medium. Next, 0.5 mL of the preculture was used to inoculate the TSB medium or modified Evans medium (30 mL). Samples were collected at different time points. Cell pellets were collected after 20 min of centrifugation at 3000 rpm. Total RNA was prepared using RNeasy Mini Kit (Qiagen, Valencia, CA). The RNA integrality was analyzed by 1% agarose gel electrophoresis and the RNA concentration was determined by microplate reader (BioTek, USA). Total RNA (1 μg) extracted from liquid cultures was reverse transcribed using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan) for real-time RT-PCR, the DNase digestion was performed to remove genomic DNA before reverse transcription for 5 min at 42 °C. All procedures above are following the manufacturer’s instructions. PCR reactions were performed with primers listed in Additional file 1 : Table S2. SYBR premix Ex Taq™ GC Kit (Perfect Real Time, Takara) was used for real-time RT-PCR, and about 100 ng cDNA was added in 20 μL volume of PCR reaction. The PCR was conducted using CFX96 Real-Time System (Bio-Rad, USA) and the PCR conditions were 95 °C for 5 min; then 40 cycles of 95 °C for 5 s, 60–64 °C for 30 s; and an extension at 72 °C for 10 min.
3
Quantitative PCR Analysis of TMEM16A Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using TRIZOL (Invitrogen Inc.). The RNA concentration was measured using GeneQuant II (Pharmacia, Uppsala, Sweden) at 260 nm. Reverse transcription reaction and cDNA synthesis was performed according to the manufacturer’s instructions (Invitrogen Inc.). Polymerase chain reaction analysis was performed on Applied Biosystems 7500 Sequence Detection system (ABI; Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq GC kit (Takara, Japan). The primers were the following: for TMEM16A, 5′-ATTTCACCAATCTTGTCTCCATCA-3′ (forward) and 5′-TGATAACTCCAAGAACGATTGCA-3′ (reverse); for GAPDH, 5′-ACACCCACTCCTCCACCTTT-3′ (forward) and 5′-TTACTCCTTGGAGGCCATGT-3′ (reverse). Gene expression of TMEM16A was normalized to the level of GAPDH within each sample using the relative ΔΔCT method. Gene expression is shown as relative expression to control. The data shown are representative of three independent experiments.
4
RNA Extraction and RT-PCR Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA preparation and real-time RT-PCR were performed as previously described (20 (link)) utilizing the primers listed in Supplementary Table S2 . Total RNA was extracted and purified from the collected cell samples using the RNeasy Mini Kit (Qiagen, Valencia, CA). About 1 μg of total RNA was reverse transcribed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan). For real-time RT-PCR, the SYBR premix Ex Taq™ GC Kit (Perfect Real Time, Takara) was used, and about 100 ng cDNA was added to a 20 μl volume of PCR reaction. The transcription values of each gene were normalized relative to the value for the internal control gene 16S rRNA (SACE_8101) using the comparative Ct method. For each gene, the relative expression value at the first time point was defined as 1. Gene expression values were determined in triplicate.
5
Gene Expression Analysis in Plants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs from isolated tissue were extracted and purified using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). 1 µg RNA was used to synthesis cDNA with the SuperScript® III Reverse Transcriptase Kit (Invitrogen, Shanghai, China). After reverse transcription, all samples were diluted with sterilized water. The reverse transcription real-time quantitative PCR (RT-qPCR) analysis was performed on the ABI 7500 Sequence Detection System (Thermo Fisher Scientific, Waltham, MA, USA) using the SYBR® Premix Ex Taq™ GC Kit (Takara, Dalian, China). Using OsACTIN1 as an endogenous control, three replicates were performed and the normalization of relative expression was calculated using the ∆∆Ct method (Livak et al. 2001 (link)). For mRNA in situ hybridization of OsCCRL1, the 331 bp specific probe of OsCCRL1 was amplified and labeled using the DIG RNA Labeling Kit (Roche, Basel, Switzerland). Pretreatment of sections, hybridization, and immunological detection were performed as described previously (Ma et al. 2017 (link)). The primer sequences used for RT-qPCR and in situ hybridization are listed in Additional file 1 : Table S1.
6
Quantification of miR-150 and PDCD4 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA in cells and tissues were extracted by using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA concentration was measured and reverse transcription reaction was performed according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.). PCR analysis was performed on Applied Biosystems 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Premix Ex Taq GC kit (Takara Bio, Inc., Otsu, Japan). The stem-loop primers used for the PCR amplification were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The relative expression level of miR-150 was normalized against U6 expression level. The primers for miR-150: 5′-CTGCTTAGTGGCTCTACTCCTG-3′ (forward) and 5′-TCCCCTCTGGCTTATGTCC-3′ (reverse); for U6: 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-TGGTGTCGTGGAGTCG-3′ (reverse); for the PDCD4: 5′-AAGAAAGGTGGTGCAGGAGG-3′ (forward) and 5′-TGACTAGCCTTCCCCTCCAA-3′ (reverse). Gene expression of PDCD4 was normalized to the level of β-actin and analyzed by the relative 2−ΔΔCq method. Each experiment was performed in triplicate.
7
Quantitative Analysis of miRNA and mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA according to the manufacturer's instructions. cDNA was reverse transcribed from total RNA using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. qPCR was subsequently performed using the SYBR Premix Ex Taq GC kit (Takara Bio, Inc.) on an ABI 7500 thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation 95˚C for 5 min, followed by 38 cycles of denaturation at 95˚C for 15 sec and annealing/elongation at 60˚C for 30 sec. The following primer pairs were used for the qPCR: miR-219-5p forward, 5'-ACACTCCAGCTGGGTGAT TGTCCAAACGCAAT-3' and reverse, 5'-CTCAACTGGTGT CGTGGA3'; LRH-1 forward, 5'-GCACGGACTTACACCTAT TGTG-3' and reverse, 5'-TGTCAATTTGGCAGTTCTGG-3'; cyclin D1 forward, 5'-AACTACCTG GACCGCTTCCT-3 and reverse, 5'-CCACTTGAGCTTGTTCACCA-3'; U6 forward, 5'-CTCGCTTCGGCAGCACATAT-3' and reverse, 5'-TTG CGTGTCATCCTTGCG-3' and GAPDH forward, 5'-CTG GGCTACACTGAGCACC-3' and reverse, 5'-AAGTGGTCG TTGAGGGCAATG-3'. GAPDH and U6 were employed as internal controls. Relative expression of genes was calculated using the 2-ΔΔCq method (17 (link)).
8
Transcriptional Analysis of M. smegmatis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mycobacterium smegmatis MC2 155 and its mutant strains were activated in LB medium (containing 0.05% tween 80) for 36–48 h at 37°C and then transferred into NXS and NL Sauton’s medium. Mid-exponential cells of M. smegmatis were collected by centrifugation (12000 rpm, 5 min, 4°C). Total RNA was obtained with the RNAprep Pure Cell/Bacteria kit (Tiangen Biotech, Beijing, China). The quality of RNA was analyzed by electrophoresis. The RNA concentrations were determined with microplate reader (BioTek). 1 μg RNA was used as template for cDNA synthesis with the PrimeScript reverse transcription (RT) regent kit (TaKaRa, Japan). The genomic DNA was removed before reverse transcription by DNase digestion for 5 min at 42°C. RT-PCR experiments were conducted with SYBR Premix Ex Taq GC kit (TaKaRa, Japan), using the primers listed in Supplementary Table S2 . PCR assays were carried out as described previously (You et al., 2017 (link)).
9
Quantification of miRNA and mRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the tissues and cells using RNeasy and miRNeasy Kits (Qiagen). One μg of total RNA was reverse-transcribed to cDNA using the Superscript II Reverse Transcriptase Kit (Invitrogen), and reverse transcription reaction were performed according to the manufacturer’s instructions. PCR was performed on an Applied Biosystems 7500 Sequence Detection System using a SYBR Premix Ex Taq GC kit (Takara). The primers were: miR-9: 5′-TCTTTGGTTATCTAGCTGTATGA-3′ (sense), 5′-TGGTGTCGTGGAGTCG-3′ (antisense); U6: 5′-CTCGCTTCGGCAGCACA -3′ (sense), 5′-AACGCTTCACGAATTTGCGT-3′ (antisense); E-cadherin: 5′-AAAGGCCCATTTCCTAAAAACCT-3′ (sense), 5′-TGCGTTCTCTATCCAGAGGCT-3′ (antisense); GAPDH: 5′-CGACCACTTTGTCAAGCTCA-3′(sense), 5′-AGGGGAGATTCAGTGTGGTG-3′ (antisense). The gene expression was normalized to the level of GAPDH using the relative ΔΔCT method.
10
Quantitative PCR of Microbial Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reverse transcription was performed with a random hexamer primer using 2 µg total RNA in a volume of 20 µl employing SuperScript III reverse transcriptase (Invitrogen). PCR was performed employing 20 ng reaction mixtures as the template, using the rpoB gene as the internal control. A negative control was performed by following the same procedures except that the addition of reverse transcriptase was omitted. Totally, 14 genes, including 6 genes related to carbon metabolisms, 4 genes related to nitrogen metabolism and 4 genes involved in rifamycin biosynthesis according to RNA-seq data, were selected for quantitative real-time PCR (qPCR) validation experiments, using a SYBR Premix Ex Taq GC kit (Takara) in a Step-one Plus real-time PCR system (Applied Biosystems).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!