Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s protocol. The RNA was reverse transcribed using ABScript II kit (Abclone; RK20402) to obtain cDNA. Real-time PCR assays were conducted and RT-PCR kit (Abclone; RK21203) and analyzed using Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Primers used are listed as followed: PRMT3 (forward: 5-GTACCCTTCTCATACCCCAATGG-3;backward:5-GACGAGCAGGTTCTGACATCT-3),HIF1α(5-GAACGTCGAAAAGAAAAGTCTCG-3;backward:5-CCTTATCAAGATGCGAACTCACA-3),VEGFA(5-AGGGCAGAATCATCACGAAGT-3;backward:5-AGGGTCTCGATTGGATGGCA-3),beta-actin(5-CATGTACGTTGCTATCCAGGC-3; backward: 5-CTCCTTAATGTCACGCACGAT-3).
Multicolor real time pcr detection system
Manufactured by Bio-Rad
Sourced in United States
The Multicolor Real-Time PCR Detection System is a laboratory instrument designed for the quantitative analysis of DNA and RNA samples using the real-time polymerase chain reaction (RT-PCR) technique. The system is capable of detecting and measuring multiple target sequences simultaneously by utilizing different fluorescent dyes or probes.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (5 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Tiangen Biotech (3 mentions)
Realstar green fast mixture, by GenStar (3 mentions)
Primer premier 5, by Premier Biosoft (2 mentions)
C1000 touch thermal cycler, by Bio-Rad (2 mentions)
Reverse transcriptase kit, by Takara Bio (2 mentions)
Starscript 2 first strand cdna synthesis mix with gdna remover, by GenStar (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Q3000 microvolume spectrophotometer, by Quawell (1 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix kit, by Bio-Rad (1 mentions)
Rnasy plant mini kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lightcycler sybr green 1 mix, by Roche (1 mentions)
25 protocols using multicolor real time pcr detection system
1
Quantifying Gene Expression in Cells
2
Validation of Saccharum SUT Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression levels of six SUT genes in three tissues (internode 9, 15 and leaf roll in LA-Purple, internode 8,13 and leaf roll in Molokai6081, and internode 6, 9 and leaf roll in SES208) of three Saccharum species were validated by qRT-PCR. Gene-specific primer pairs were designed by using Integrated DNA Technologies (IDT) (http://www.idtdna.com/Primerquest/Home/Index ). After treated with DNase I (Tiangen, China), two microgram of RNA was used in reverse transcription with the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s guidelines. The real-time qPCR was performed by using Multicolor Real-Time PCR Detection System (Bio-Rad) with conditions for all reactions were 95 °C for 30s, 40 cycles of 95 °C for 5 s, followed by 60 °C for 30s, and 95 °C for 10s. Melting curve analysis were performed to confirm the PCR specificity. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and Eukaryotic elongation factor 1a (eEF-1a) were selected as internal standard for normalization [39 (link)], and three replicates were completed for each sample. The relative expression level for each SUT gene in different tissues of three Saccharum species were calculated by using the 2-ΔΔCt method. The correlation coefficient was calculated between the transcript accumulation levels obtained by RNAseq and qRT-PCR using Excel.
3
Quantifying NLRP3 Inflammasome Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol reagent (Applied Biosystems, Waltham, MA, United States) was used to extract total RNA, and cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, United States). The primers for NLRP3, ASC, and CASPASE-1 mRNA sequence used were as described in NCBI (shown in Table 1 ) and were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). SYBR®Green PCR Master Mix (Applied Biosystems) was used as the qRT-PCR reaction system with a volume of 25 µl and detected using a Multicolor Real-time PCR detection system (Bio-Rad, Hercules, CA, United States). The testing conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 60°C for 1 min. The 2−ΔΔCt method was used to calculate relative mRNA expression in each sample.
4
Lipid Metabolism Pathway Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six unigenes involved in different lipid metabolism pathways with different regulation modes were selected for the validation using real time qPCR. The gene-specific primer pairs were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). Total RNA was isolated from fruits sampled at 10, 80, 140, and 170 DAF as the description mentioned above. cDNA was synthesized using the SYBR Premix Ex Taq Kit (TaKaRa, Mountain View, CA, USA) according to the manufacturer’s protocol. Relative mRNA abundance of the selected genes was determined using Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The actin-related protein 3 (ACTR3) and 18S rRNA were chosen as an internal control for normalization. The conditions for all reactions were 95 °C for 2 min, 40 cycles of 95 °C for 15 s, followed by 60 °C for 15 s, and 95 °C for 15 s. Three technical repetitions were performed for qRT-PCR. The relative expression level of qPCR results for each unigene was calculated using the comparative cycle threshold (ΔΔCt) method [34 (link)]. Correlation analysis between RPKM with ΔΔCt was performed using JMP (Version 12, SAS Institute Inc., Cary, NC).
5
Quantitative RT-PCR for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
Realtime quantitative PCR was carried out in a 25 µL reaction mix containing 12.5 µL of SYBR Premix Ex Taq (TaKaRa, (
www.takara.com.cn ), 1 µL of 1:5 diluted cDNA template, 1 µL of each of the primers (10 µM), and 9.5 µL ddH2O. The thermal cycling profile consisted of initial denaturation at 95°C for 30 sec and 40 cycles at 95°C for 5 sec, 60°C for 30 sec, and 72°C for 20 sec. Relative expression levels were calculated using the 2−∆∆Ct method, where ∆∆Ct=∆Ctsample−∆Ctreference following the previously published protocol (
Livak and Schmittgen 2001 (link)
). PCR reactions were performed in 96-well plates with a Multicolor Realtime PCR Detection System (Bio-Rad, (
www.bio-rad.com ) using SYBR Green to detect dsDNA synthesis. PCR amplification was performed in duplicate wells.
+ Expand
Realtime quantitative PCR was carried out in a 25 µL reaction mix containing 12.5 µL of SYBR Premix Ex Taq (TaKaRa, (
Livak and Schmittgen 2001 (link)
). PCR reactions were performed in 96-well plates with a Multicolor Realtime PCR Detection System (Bio-Rad, (
6
Hippocampal TPH2 and IDO1 mRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The levels of TPH2 and IDO1 mRNAs in the hippocampus were evaluated by qRT-PCR assay. The total RNA of hypothalamus was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Then, the concentration and quality of total RNA were determined by Q3000 micro-volume spectrophotometer (Quawell Technology, San Jose, CA, USA) and 1% agarose gel electrophoresis. The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) was used to synthesize first-strand cDNA on C1000 Touch TM Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the standard protocol. The SYBR® Green PCR Master Mix (Thermo Fisher Scientific) was used to amplify the cDNA in the Multicolor Real-time PCR Detection System (Bio-Rad Laboratories Inc.), and the cycling parameters were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 56°C (TPH2) or 58°C (IDO1) for 1 minute, then followed by 65°C for 5 seconds and 95°C for 15 seconds. The 2−∆∆Ct method was used to calculate the expression of mRNA. Table 1 shows the sequences for primers, which were designed according to the published mRNA sequences in NCBI, and GAPDH (BBI Life Science, Amherst, MA, USA) was used as the house-keeping gene in the experiment.
7
Quantifying Elven Unigenes in Tea
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Elven important unigenes potentially involved in some of the important secondary metabolites biosynthesis pathways were selected for qRT-PCR experiments. Gene-specific primer pairs were designed using Primer primer 5.0 software (Premier Biosoft International), and total RNA was isolated from prepared tea samples using a modified CTAB method, respectively. After treated with DNase I (Tiangen, China), one microgram of RNA was used in reverse transcription with the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s guidelines. The standard curve for each gene was conducted in several dilutions of cDNA, then real-time qPCR was performed using Multicolor Real-Time PCR Detection System (Bio-Rad) with conditions for all reactions were 95 °C for 10 min, 40 cycles of 95 °C for 15 s, followed by 60 °C for 30 s. Melting curve and agarose gel electrophoresis analysis were performed to confirm the PCR specificity. The 18S RNA gene was selected as an internal standard for normalization, and three biological replicates were completed for each gene. The relative expression levels for each unigene were in the different tissues calculated by using the delta-delta Ct (2-ΔΔCt)method. All data were expressed as the mean ± SD after normalization.
8
Quantitative RT-PCR of ABCG2, BAX, and BCL2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The quantitative reverse transcription PCR of ABCG2 and BAX and BCL2 was performed according to the SYBR green protocol (Amplicon, Denmark). 25 μL of reaction volume contains 20 ng of cDNA and 0.5 Μl of ABCG2 specific primers (Table 1 ) and 13 μL of Real Q Plus 2× Master Mix and 6 μL of PCR-grade H2O. Reactions including ABCG2 gene and β2M gene as reference genes were analyzed and run on a multi-color real-time PCR detection system (Bio-Rad, USA). Average Ct triplet repeats were used for data analysis. The cycling parameters were analyzed and determined using statistical analysis. The integrity of real-time qPCR products were confirmed by separating on a 2% agarose gel.
9
Quantifying GFAP and NR2B mRNA Changes in Hippocampus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Next, we continue to explore changes in GFAP and NR2B mRNAs in the hippocampus. The total RNA in the hippocampus of each rat was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of total RNA was determined by a spectrophotometer (Eppendorf, Germany), and the purity of RNA was measured by 1% agarose gel electrophoresis. The RNA from each sample was utilized to synthesize the first-strand cDNA using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) on a C1000 TouchTM Thermal Cycler (Bio-Rad, California, CA, USA). The sequences for primers showed in Table 2
10
qRT-PCR Analysis of FRK Genes in Sugarcane
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA (≤1 μg) from each sample was reverse-transcribed to cDNA using the Reverse Transcriptase Kit (Takara) in a 20 μl reaction volume with 1 μl of random primers and 1 μl of mixed poly-dT primers (18–23 nt). The cDNA was diluted 1:7 in water for further qRT-PCR experiments.
The expression levels of FRK genes were validated using qRT-PCR in partial tissues of four sugarcane species. Gene-specific primer pairs (Additional file9 ) were designed using the online PrimerQuest tool at Integrated DNA Technologies (IDT) (http://www.idtdna.com/Primerquest/Home/Index ). Real-time qPCR were run on Multicolor Real-Time PCR Detection System (Bio-Rad). The real time PCR reaction program was 95 °C for 30s, 40 cycles at 95 °C for 5 s, followed by 60 °C for 30s, and PCR specificity was confirmed using a heat dissociation protocol from 65 to 95_C following the final cycle of the PCR. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and Eukaryotic elongation factor 1a (eEF-1a) were selected as internal standards for normalization [47 (link)], and three replicates were run for each sample. The relative expression levels for each FRK gene in different tissues of three sugarcane species were calculated using the 2-ΔΔCt method [48 (link)].
The expression levels of FRK genes were validated using qRT-PCR in partial tissues of four sugarcane species. Gene-specific primer pairs (Additional file
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!