Engineered S. thermophilus was grown on LM17 medium for 24 h and collected by centrifugation (12,000 × g, 2 min, 4°C). Total RNA was isolated using the RNAiso Plus kit (Takara Technology Co., Ltd.) according to the manufacturer's instructions. Total RNA was reverse-transcribed using the PrimeScript RT reagent kit with gDNA Eraser (Takara). We used 100 ng of cDNA for gene expression analysis. Real-time quantitative (RT-qPCR) was performed in triplicate using the Lightcycler 96 RT-qPCR system (Roche Diagnostics). Data were analyzed using Lightcycler 96 software and the 2 -ΔΔCt method with recA as a reference gene (Kong et al., 2019) (link).
Lightcycler 96 rt qpcr system
Manufactured by Roche
Sourced in Switzerland, Germany
The LightCycler 96 RT-qPCR system is a real-time PCR instrument designed for quantitative analysis of nucleic acid samples. It supports multiple detection chemistries and allows for fast and precise quantification of target sequences. The system provides a reliable and efficient platform for a wide range of molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Cw0584s, by CWBIO (2 mentions)
Cw0659, by CWBIO (2 mentions)
Cw2020m, by CWBIO (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Total rna midi kit, by Omega Bio-Tek (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Tb green premix ex taq 2 til rnaseh plus, by Takara Bio (1 mentions)
High capacity cdna reverse transcription kit, by Promega (1 mentions)
Power sybr green master mix, by Takara Bio (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Sybr green pcr kit, by Vazyme (1 mentions)
Superscript 2 reverse transcription kit, by Vazyme (1 mentions)
9 protocols using lightcycler 96 rt qpcr system
1
Quantifying S. thermophilus Gene Expression
2
Transcriptomic Analysis of Streptococcus thermophilus ST-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Streptococcus thermophilus ST-1 was activated in LM17 overnight cultures, then transferred to CDM with 0.5 mM GSH at a dilution of 1:50. Cells were collected from CDM when the OD 600nm reached 0.5. Total RNA was isolated with a total RNA midi kit (Omega Bio-Tek Inc., Norcross, GA), and the genomic DNA was eliminated using DNase I (Takara). The quantification and integrity of purified RNA were assessed using Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and agarose gel electrophoresis. The cDNA was prepared by PrimeScript reverse-transcriptase (RT) reagent kit with gDNA Eraser (Takara) and used as template for RT quantitative PCR (qPCR), which was performed on a new Lightcycler 96 RT-qPCR system (Roche Diagnostics, Rotkreuz, Switzerland) using a SYBR Premix Ex TaqII kit (Takara). The prim-ers of RT-qPCR are listed in Supplemental Table S1 (https: / / doi .org/ 10 .3168/ jds . 2020 -19234) . The 2 -ΔΔCt method was used to relatively quantify the expression differences of the target genes in Strep. thermophilus ST-1 under static culture or aerobic culture conditions with Lactobacillus CDM exogenous addition of GSH (Schmittgen and Livak, 2008) (link). The reference conditions were the expression of the same gene in Lactobacillus CDM without exogenous addition of GSH, and the 16S rDNA gene of ST-1 was selected as the housekeeping gene (Wang et al., 2015a) .s
3
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted with RNAiso Plus (#9109, TakaRa Bio Inc, Shiga, Japan) as previously described. RNA was reverse transcribed into cDNA with PrimeScript RT reagent Kit (#RR037, TakaRa Bio Inc). cDNA was then amplified with TB green Premix Ex Taq II (Til Rnase H Plus) (##RR820A, TakaRa Bio Inc). The real-time PCR was conducted with a LightCycler 96 RT-qPCR System (Roche, Basel, Switzerland). The relative quantity of the targeted RNA was calculated through normalization to the quantity of the corresponding GAPDH mRNA level. Detailed primer sequences are listed in Supplementary file 2 .
4
Quantitative Analysis of Liver Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from liver tissues with Trizol Reagents (Life technologies, Carisbad, CA, USA). The RNA was reverse-transcribed into complementary (c) DNA using a high-capacity cDNA reverse transcription kit (Promega Biotech Co., Ltd., Madison, WI, USA). Genes of PPARα, SREBP1, toll like receptor (TLR)-4, MyD88, IκBα, and NOS2 were amplified through real time-quantitative polymerase chain reaction (RT-qPCR) with the primers shown in Table 1 . GAPDH was used as internal control in all reactions. A LightCycler® 96 RT-qPCR system (Roche, Mannheim, Germany) was employed, with 20 μL reaction volume containing 12.5 μL POWER SYBR green master mix (TAKARA, Dalian, China), 5 μmol/L primer, and 25 ng cDNA template. Reaction was performed with an initial hold step at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. Melting curves of PCR products were acquired stepwise from 55 to 95 °C to ensure their purity. Comparative CT method was employed for target gene quantification, and to be normalized to GAPDH and relative to a calibrator (2−ΔΔCt).
5
RT-qPCR Assay for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RT-qPCR was performed using QIAGEN OneStep RT-PCR Kit (QIAGEN, Hilden, Germany) with SYTO 9 (Life technologies, Carlsbad, CA, USA) as the fluorescent dye. Thirty μL reactions including 3 μL template input were run on a Light Cycler 96 RT-qPCR System (Roche Diagnostics, Mannheim, Germany). Reaction conditions were: 30 min RT at 50 °C, 15 min at 94 °C for inactivation of reverse transcriptase (RT), followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min. Melting curve analysis was performed under the condition of 95 °C for 60 s, 40 °C for 60 s, 65 °C for 1 s, then followed by a slow increase from 65 °C to 95 °C with a speed of 0.07 °C per second.
6
Quantitative RT-PCR Analysis of Colon Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA of colon tissues was extracted using a commercial kit (CWBiotech; CW0584S), and RNA concentration and quality were evaluated with an ultra-micro spectrophotometer (NanoDrop 2000; Thermo Fisher) and 1% agarose gel electrophoresis. One thousand nanograms of RNA was reverse transcribed to cDNAs using a HiFiScript reverse transcriptase mixture with genomic DNA remover (CWBiotech; CW2020M). The SYBR green (CWBiotech; CW0659) method was used to quantify gene expression levels with a real-time quantitative PCR (RT-qPCR). The 10-μL mixture including 0.2 μM primer and 2 μL diluted cDNAs was run on the LightCycler 96 RT-qPCR system (Roche), followed by predenaturation at 95°C for 60 s and 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 45 s. GAPDH was used as an internal reference. All the primers for target genes are listed in Table S3. Standard and dissolution curves were obtained to confirm efficiency and specificity of the primers. The 2−ΔΔCT method was used to calculate relative expression level of target genes.
7
Quantitative RT-PCR Analysis of Colon Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA of colon tissues was extracted using a commercial kit (CWBiotech; CW0584S), and RNA concentration and quality were evaluated with an ultra-micro spectrophotometer (NanoDrop 2000; Thermo Fisher) and 1% agarose gel electrophoresis. One thousand nanograms of RNA was reverse transcribed to cDNAs using a HiFiScript reverse transcriptase mixture with genomic DNA remover (CWBiotech; CW2020M). The SYBR green (CWBiotech; CW0659) method was used to quantify gene expression levels with a real-time quantitative PCR (RT-qPCR). The 10-μL mixture including 0.2 μM primer and 2 μL diluted cDNAs was run on the LightCycler 96 RT-qPCR system (Roche), followed by predenaturation at 95°C for 60 s and 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 45 s. GAPDH was used as an internal reference. All the primers for target genes are listed in Table S3. Standard and dissolution curves were obtained to confirm efficiency and specificity of the primers. The 2−ΔΔCT method was used to calculate relative expression level of target genes.
8
qRT-PCR Analysis of CENPO Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol reagent (Invitrogen) was used to extract RNA from tissues or cells, and the concentration and purity of the extracted RNA was determined using a NanoDrop 2000 (Thermo Scientific). Reverse transcription of extracted RNA into cDNA was conducted using a Superscript II Reverse Transcription Kit (Vazyme Biotech, Nanjing, China). Real-time PCR assays were conducted on a LightCycler® 96 RT-qPCR system (Roche, Switzerland) using SYBR Green PCR Kits (Vazyme Biotech, Nanjing, China). GAPDH served as the endogenous control. The primers used in this investigation were synthesized by Sangon Biotechnology (Shanghai, China), as follows: CENPO, Forward, 5’-TTACAGACCAACATCCAGCACTTCC-3’, Reverse, 5’-GCAAGGGCTTCGTACAGAACAGAG-3’; and GAPDH, Forward, 5’-AAGGTCATCCCTGAGCTGAA-3’, Reverse, 5’-TGACAAAGTGGTCGTTGAGG-3’. PCR amplification conditions: 95℃, 15 sec; 95℃, 10 sec; 60℃, 30 sec, 40 cycles. Results were analyzed by calculating the relative expression of CENPO mRNA using the 2−ΔΔCt method.
9
Quantifying mRNA Levels via RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the cells treated for 24 h using an RNeasy Mini Kit (74104; Qiagen GmbH, Germany) according to the manufacturer's protocol. Subsequently, 1 μg of isolated RNA was reverse transcribed into cDNA using HiScript II QRT SuperMix (KCD-M1003, Croda Co. Ltd., China). Next, cDNA was analyzed via qPCR using SYBR qPCR Master Mix (KCD-M1004; Croda Co. Ltd., China) in a LightCycler 96 RT-qPCR system (Roche Group, Switzerland). The relative mRNA expression levels were quantified using the 2 -ΔΔCt method, with GAPDH serving as the reference gene. The primers used were synthesized by Jiangsu Saisofei Biotech Co., Ltd. (Wuxi, China). The primer sequences are shown in supplementary Table 1.
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!