The RNA samples were treated with RQ1 RNase-Free DNase (Promega, USA) to eliminate residual genomic DNA and reverse transcribed using the PrimeScript® RT Reagent Kit (TaKaRa, Japan), and the qRT-PCR reactions were performed on a Bio-Rad CFX96 Real-Time PCR System using the SYBR Green EX Taq mix (TaKaRa, Japan) with the kit-recommended two-step standard cycling method. Every qRT-PCR was performed in triplicate with a volume of 25 μL and repeated more than three times under the same conditions in a separate experiment. For the relative quantification of specific genes from different transcripts, the cycle threshold (CT) method was used to calculate fold changes in expression. The 16S rRNA gene was chosen as the reference gene to normalise the variability in expression levels. The orf_1053, orf_1589, orf_2472 and orf_2475 genes were selected to verify the RNA sequencing results. Primer Premier 5 was used to design the forward and reverse primers for each selected gene. The efficiency of each primer pair was calculated using the standard curves of 10-fold serial dilutions of genomic DNA by the Bio-Rad CFX Manager.
Sybr green ex taq mix
Manufactured by Takara Bio
Sourced in Japan
SYBR Green EX Taq mix is a ready-to-use solution for real-time PCR amplification. It contains SYBR Green I dye, Taq DNA polymerase, dNTPs, and buffer components optimized for sensitive and efficient detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Cfx96 real time pcr system, by Bio-Rad (2 mentions)
Cfx 96 real time pcr system, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Cfx manager, by Bio-Rad (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
4 protocols using sybr green ex taq mix
1
qRT-PCR Analysis of Gene Expression
2
Salt Stress-Response Genes Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We selected 53 salt stress-response genes encoding sigma factor, glutamate biosynthesis, peptidoglycan biosynthesis and ion-transport proteins. Specific primers for each gene were designed using primer 3 batched run by a Perl script [13 ] and 109–168 bp products were amplified (see Additional file 2 : Table S2 for primer details).
Fluorescence PCR amplifications were performed using SYBR Green EX Taq mix (TaKaRa) in a Bio-Rad CFX96 Real-Time PCR system. The relative transcript level of each gene (cultures cultivated in LB broth containing 5% NaCl) was determined by calculating 2-ΔΔct compared with the transcript level of control sample (cultures cultivated in LB broth containing 0.5% NaCl) using gyrB or thyA gene as internal control. ddH2O was used as negative control for qRT-PCR. These genes were then used for GO term functional analysis and KEGG pathway analysis. Each experiment was conducted in triplicates.
Fluorescence PCR amplifications were performed using SYBR Green EX Taq mix (TaKaRa) in a Bio-Rad CFX96 Real-Time PCR system. The relative transcript level of each gene (cultures cultivated in LB broth containing 5% NaCl) was determined by calculating 2-ΔΔct compared with the transcript level of control sample (cultures cultivated in LB broth containing 0.5% NaCl) using gyrB or thyA gene as internal control. ddH2O was used as negative control for qRT-PCR. These genes were then used for GO term functional analysis and KEGG pathway analysis. Each experiment was conducted in triplicates.
3
Quantification of HPV E6 and E7 mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the HeLa cells using TRIzol, following the manufacturer's instructions. The isolated RNA was reverse transcribed into complementary DNA (cDNA) using the PrimeScript RT Master Mix (Takara, Japan) under the following thermal cycling conditions: 37°C for 30 min and 85°C for 5 s and hold at 4°C. The qRT-PCR analysis was performed using the SYBR Green Ex Taq mix (Takara, Japan), following the manufacturer's instructions. Each sample was analyzed in triplicate. The PCR conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, and 60°C for 45 s. The mRNA levels of E6 and E7 were quantified using qRT-PCR. GAPDH and beta-actin were used as an internal control. The following primers were used for qRT-PCR analysis: HPV type 18 E6, 5′-ATAAGGTGCCTGCGGTGCC-3′ (forward) and 5′-TGCGTCGTTGGAGTCGTTC-3′ (reverse); HPV type 18 E7, 5′-GAGCACGACAGGAACGACT-3′ (forward) and 5′-GGGCTGGTAAATGTTGATGAT-3′ (reverse); GAPDH, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (forward) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ (reverse); beta-actin, 5′-GACGACATGGAGAAAATCTG-3′ (forward) and 5′-ATGATCTGGGTCATCTTCTC-3′ (reverse). The fold change of target mRNA expression was analyzed with the 2-ΔΔCt method.
4
Quantitative RT-PCR Analysis of S. algae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Superscript III first-strand synthesis system (Invitrogen) was used to generate cDNA using 1 µg of RNA and oligo dT primer, according to the manufacturer’s instructions. The qRT-PCR amplifications were performed using the SYBR Green EX Taq mix (TaKaRa) on a Bio-Rad CFX96 Real-Time PCR system. The relative expression level of the specific genes were determined by calculating 2−ΔΔcq compared with the expression level of 0 h time point using 16s rRNA gene as an internal control. The qRT-PCR reactions were performed in triplicate for two biological replications. The specific primers for each gene see Table S1 .
Standard curves were generated for each gene to evaluate primer efficiency and for data analysis. S. algae strain 2736 was cultivated in biological triplicate in LB broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) for 5 h (OD600 = 1.6) then total RNA was extracted, digested and converted to cDNA. Using 10-fold serial dilutions of the cDNA templates, qRT-PCR amplifications were performed. The standard curve was generated by plotting the Ct values versus ten times serial dilutions of the RNA templates ranging from 2.5×100–2.5×10−4 ng/µl.
Standard curves were generated for each gene to evaluate primer efficiency and for data analysis. S. algae strain 2736 was cultivated in biological triplicate in LB broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) for 5 h (OD600 = 1.6) then total RNA was extracted, digested and converted to cDNA. Using 10-fold serial dilutions of the cDNA templates, qRT-PCR amplifications were performed. The standard curve was generated by plotting the Ct values versus ten times serial dilutions of the RNA templates ranging from 2.5×100–2.5×10−4 ng/µl.
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!