In the present study, 10-fold serial dilutions of RNA molecular standard ranging from 5.2 × 108–5.2 × 100 RNA molecules per reaction were prepared for the standard curve. Real-time RT-PCR was performed on a Light Cycler 480 Real-Time PCR System using One Step TB Green® PremixScript™ RT-PCR kit (Takara Bio Inc., Japan). Briefly, after a reverse transcription at 42 °C for 5 min, and 95 °C for 10 s, amplifications were carried out with 40 cycles at a melting temperature of 95 °C for 5 s and an annealing temperature of 60 °C for 20 s, followed by a melting curve analysis under conditions as follows: 95 °C for 5 s, 65 °C for 60 s, and 95 °C for 15 s [51 (link)]. The standard curve was generated using 10-fold-serial dilutions of molecular standard and threshold cycle (Ct) values. The Ct values were compared between each of them, and the sensitivity of this method from the standard curve was determined. Moreover, the presence of non-specific PCR amplification could be ruled out by performing a melting curve analysis.
Lightcycler 480 real time pcr system
Manufactured by Takara Bio
Sourced in Japan
The LightCycler 480 Real-Time PCR system is a laboratory instrument designed for performing real-time quantitative polymerase chain reaction (qPCR) analysis. It is capable of detecting and quantifying nucleic acid sequences in real-time, allowing for accurate and sensitive analysis of DNA and RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Sybr premix ex taq, by Takara Bio (3 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Tb green premix ex taq 2, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Minibest plant rna extraction kit, by Takara Bio (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Tb green premix ex taq tli rnaseh plus, by Takara Bio (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Sybr premix ex taqtm kit, by Takara Bio (1 mentions)
Primescript rt reagent kit with cdna eraser, by Takara Bio (1 mentions)
One step tb green primescript rt pcr kit perfect real time, by Takara Bio (1 mentions)
Mir x mirna qrt pcr sybr kit, by Takara Bio (1 mentions)
Sybr premix ex taq tli rnaseh plus kit, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Sybr green reagent, by Takara Bio (1 mentions)
First strand cdna synthesis kit, by Takara Bio (1 mentions)
18 protocols using lightcycler 480 real time pcr system
1
Real-Time RT-PCR Quantification of RNA Molecules
2
Quantifying JcHDZ16 Expression in Rice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To estimate JcHDZ16 expression levels in wild type and transgenic rice, total RNA was isolated from selected organs of 2-week-old seedlings that had been sampled and stored at − 80 °C, using a MiniBEST plant RNA extraction kit (TaKaRa Code No. 9769). 2 μg RNA samples were used to synthesize first-strand cDNA using M-MLV reverse transcriptase (Promega, http://www.promega.com ). JcHDZ16 sequences were then quantified by qRT-PCR using SYBR GreenSYBR® Premix Ex Taq™ (TaKaRa, Japan) and a LightCycler® 480 real-time PCR system (http://www.roche.com/ ), with the following settings: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, 60 °C for 20 s, and 72 °C for 20 s. The cited manufacturers’ instructions were followed in all of these procedures. Relative expression levels were calculated using the 2-∆∆CT method, and JcActin and Osubiquition as reference genes for physic nut and rice, respectively. All primers used in this study are listed in Additional file 7 . Three biological replicates and two technical replicates of each biological replicate were used in this experiment.
3
Quantitative Analysis of Sterol Synthesis Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was reverse transcribed into cDNA using the Integrated First Strand cDNA Synthesis Kit (DiNing, Beijing, China). The expression levels of unigenes putatively involved in sterol synthesis were monitored by RT-qPCR using the primer sequences listed in Table S1 (Supplementary Table S8 ). RT-qPCR analysis was performed with a Roche LightCycler480 Real-time PCR System using SYBR Premix Ex Taq (TaKaRa, Tokyo, Japan). The reaction conditions were as follows: 95 °C for 10 min, and 40 cycles of 95 °C for 15 s, 58 °C for 1 min. The actin (ACT) gene was used as an internal reference, and the relative expression level of each unigene was determined using the 2−ΔΔCt method53 (link).
4
Quantitative Real-Time PCR of Mitochondrial Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative real-time PCR (RT-PCR) was performed on HSECs and clinical samples. β-actin was used as a reference for normalization. Nuclear respiratory factor 1 (NRF1), nuclear respiratory factor 2 (NRF2), transcription factor A, mitochondrial (TFAM), and cytochrome c oxidase subunit 4 (COX4) PCR was performed with the Roche LightCycler 480 Real-Time PCR System using SYBR Premix Ex Taq (Takara). The primer sequences can be found in Supplementary Table 1 . Relative gene expression was calculated using the 2−ΔΔCT method.
5
Quantitative Real-Time PCR Analysis of Intestinal Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Basal-out organoids and apical-out organoids were collected after experiments. Total RNA was extracted from intestinal organoids and L. fermentum (FL4) strain by Trizol assay. RNA was further reverse-transcribed into cDNA using PrimeScript RT Master Mix (Perfect Real Time) (Takara) following the manufacturer’s instructions. All cDNA were analyzed on LightCycler 480 Real-time PCR System with the TB green Premix Ex Taq (Tli RNaseH Plus, Takara) and specific primer (Table 1 ). Reaction conditions: 95 °C, 5 s; 40 cycles of 95 °C, 5 s and 60 °C, 30 s; 95 °C, 5 s; 60 °C, 1 min; 95 °C, 5 s; 50 °C, 30 s. GAPDH was used as endogenous gene for porcine intestinal organoids, while phenyl alanyl synthase (pheS) was used as control gene for L. fermentum. Assays were performed in triplicate, and the relative expression of target gene was analyzed based on the 2−ΔΔCt method.
6
RNA-seq Differential Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein-coding gene expression levels in each sample were estimated according to fragments per kilo-base of exon per million fragments mapped and assessed with the analysis software Cufflinks v2.1.1 (Ghosh and Chan, 2016 (link)). P adjusted values were calculated using the Benjamini-corrected modified Fisher's exact test, Transcripts with a P adjusted value < 0.05, |fold change| > 1.5, were considered differentially expressed. The results of RNA sequencing were validated through qPCR. qPCR was performed using the LightCycler 480 real-time PCR system and SYBR Green PCR Master Mix (TaKaRa Biotechnology, Dalian, China). Total RNA was isolated from the ileac tissues using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). cDNA was generated using the PrimeScript RT reagent kit with cDNA eraser (Takara, Dalian, China). The specific quantitative primers for 7 transcripts are listed in Supplementary Data 2 . Quantitative PCR assays were carried out using the SYBR Premix Ex TaqTM kit (Takara, Dalian, China). The conditions were 95°C for 2 min followed by 40 cycles (95°C for 20 s, 60°C for 30 s, and 68°C for 30 s). Each experiment was performed in triplicate. Target gene expression was quantified using the 2-△△CT method and normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase.
7
Analysis of 12 miRNA Expression in Breast Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We totally collected 60 BC samples from patients and 30 normal breast tissues who underwent surgical treatments in Shengjing Hospital of China Medical University from 2007 to 2021. In these 60 BC samples, they were HR-, HER2+++ on immunohistochemistry or HR-, HER2++ on immunohistochemistry with fluorescence in situ hybridization (FISH) indicating that HER2 was amplified. Formalin fixation and paraffin embedding (FFPE) were to preserve the specimens. The study was approved by the hospital institutional ethics review committee. For evaluating the expression levels of 12 miRNA, we deparaffinized these specimens using xylene and ethanol. According to the manufacturer’s protocol, we extracted total RNA (including miRNAs) from FFPE tissue samples using TRIzol (Thermo Fisher Scientific, US), and cDNA synthesis was carrying out by using Mir-X miRNA qRT-PCR SYBR Kits (Takara Bio Inc., Kusatsu, Japan). Then, we performed real-time PCR reaction using One Step TB Green® PrimeScript™ RT-PCR Kit (Perfect Real Time) (Takara Bio Inc., Kusatsu, Japan) on The LightCycler 480 Real-Time PCR System. 12 miRNAs expression levels were calculated by the 2-ΔΔCt method and the cycle threshold (CT) values of miRNAs were normalized to the level of U6 as internal reference. Primers sequences used in our study were shown in table (Supplementary Table S2
8
Quantifying eIF3a and HE4 mRNA Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from frozen patient tissues using the Trizol reagent (Thermo Fisher). RNA was reverse transcribed using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara), and RT-qPCR was carried out in a LightCycler® 480 Real-Time PCR System using the SYBR Premix Ex Taq™ (Tli RNaseH Plus) kit (Takara), according to the manufacturers' instructions. The primers were as follows: for eIF3a, 5'-TGATGAGGACAGAGGACCAAGAC-3'(forward) and 5'-TCAGCATTACGCCAGGATGA-3' (reverse); for HE4, 5'-ATAGCACCATGCCTGCTTGT-3' (forward) and 5'-TGCTCCTGTGCCTGAGACTA-3'(reverse); and for GAPDH control, 5'-GAAATCCCATCACCATCTTCCAGG-3' (forward) and 5'-GAGCCCCAGCCTTCTCCATG-3' (reverse). GAPDH acted as the internal control. The relative mRNA levels of HE4 and eIF3a were defined by using the 2-△△Ct cycle threshold method.
9
Quantitative Analysis of B7-H3 mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from homogenized frozen tissues using TRIzol reagent (Guangzhou IGE Bio.) and reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit (Guangzhou IGE Bio.) according to the manufacturer’s instructions. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was performed on a Roche LightCycler 480 Real-Time PCR system using a SYBR Premix Ex Taq (Takara) according to the manufacturer’s instructions. The reaction protocol was as follows:1 min at 95°C, 40 cycles of 10 seconds at 95°C and 12 min at 60°C, and finally a 65°C to 95°C ramp up to determine the melting curve. Relative mRNA level of B7-H3 was normalized to that of GAPDH mRNA. The relative mRNA expression was calculated using the comparative 2-△△Ct method.
10
RNA Extraction and RT-qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells or tissues using RNAiso Plus (Takara D9108, Dalian, Liaoning, China) following the instructions from the manufacturer. RNA concentration was determined by Nanodrop. Reverse procedure of cDNA was done using First-strand cDNA Synthesis kit (Takara RR037A). RT-qPCR was performed using the Roche Lightcycler480 Real-time PCR System with SYBR green reagents from Takara (RR820A). Genes were amplified using the primers described in Supplementary Table . Quantifications were normalized to GAPDH for cell or 18 s rRNA for tissue.
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!