MC3T3-E1 cells were grown in a differentiation-inducing medium for 24 hours, and total RNA was isolated using Beyozol reagent (Beyotime). The RNA integrity was determined by separating the RNA on an agarose gel and quality was assessed by measuring the A260/A280 and A260/A230 ratio cutoff higher than 1.8 and 2.0, respectively. Two micrograms of total RNA were used for reverse transcription to synthesize cDNA with dNTP mix and RNase-free H2O, following the manufacturer's protocols. qRT-PCR was performed using SYBR1 Premix Ex TaqTM (Takara, Dalian, Japan). The qRT-PCR parameters were as follows: 95.0°C for 30 s, 39 cycles of 95.0°C for 5 s and 60.0°C for 30 s, and 95.0°C for 10 s, followed by a melt curve from 65.0 to 95.0°C at an increment of 0.5°C per 5 s. Three samples were obtained from three independent experiments, and each sample was analyzed in triplicate. The gene expression data were normalized to an internal control (GAPDH) and were analyzed using the comparative 2-ΔΔCq method only when amplification efficiency calculated using the standard curve of the mRNAs of interest and the internal mRNA was 90%~110% 32 (link). GraphPad Prism 7 was used to calculate the data as fold changes compared with the control group. The primer sequences used are shown in Table 1 .
Beyozol reagent
Manufactured by Beyotime
Sourced in China, Japan
Beyozol is a reagent used in various laboratory applications. It is a colorless, water-soluble liquid with a neutral pH. Beyozol's core function is to facilitate chemical reactions and analyses in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt master mix, by Takara Bio (3 mentions)
Sybr1 premix ex taqtm, by Takara Bio (2 mentions)
Lightcycler rna master sybr green 1, by Roche (2 mentions)
Beyofast sybr green qpcr mix kit, by Beyotime (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Prism 7, by GraphPad (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Beyort first strand cdna synthesis kit, by Beyotime (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Sybr1 premix ex taq, by Takara Bio (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Forget me not qpcr master mix, by Biotium (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Beyort 3 first strand cdna synthesis kit, by Beyotime (1 mentions)
15 protocols using beyozol reagent
1
Gene Expression Analysis of MC3T3-E1 Cells
2
Quantitative Analysis of circRERE and miR-152-3p Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As per the instruction book, Beyozol reagent (Beyotime, Shanghai, China) was used for the RNA purification. RNA was then reversely transcribed into the complementary RNA (cDNA) by the miScript II RT Kit and QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The quantitative expression was detected using miScript SYBR Green PCR Kit and QuantiTect SYBR® Green PCR Kit (Qiagen), followed by the data analysis by the 2−ΔΔCt method [17] (link). The sequences for all primers were presented as below: circRERE, 5′-TTTGCAGGAATGTGTGATGG-3′ (forward, F) and 5′-ATGAATCCACTCGGGCTTTA-3′ (reverse, R); RERE, 5′-GCAGAGACAGTGAAGAAGTCGG-3′ (F) and 5′-CTTCTTGGAGCTGGTCCTGTCA-3′ (R); miR-152-3p, 5′-GCGCTCAGTGCATGACAGA-3′ (F) and 5′-GTCGTATCCAGTGCAGGGTC-3′ (R); CD47, 5′-TATCCTCGCTGTGGTTGGACTG-3′ (F) and 5′-TAGTCCAAGTAATTGTGCTAGAGC-3′ (R); glyceraldehyde-phosphate dehydrogenase (GAPDH), 5′-GTCTCCTCTGACTTCAACAGCG-3′ (F) and 5′-ACCACCCTGTTGCTGTAGCCAA-3′ (R); U6, 5′-CTCGCTTCGGCAGCACA-3′ (F) and 5′-AACGCTTCACGAATTTGCGT-3′ (R). The normalization of expression level was performed by using GAPDH and U6 as the internal controls.
3
Quantifying Gene Expression in Colon Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of colon tissues was extracted employing Beyozol reagent (R0011, Beyotime, China) following the manufacturer’s instruction. A BeyoRT™ First Strand cDNA Synthesis Kit (D7166, Beyotime, China) was utilized to produce the complementary DNA according to the protocol of the kit. Then the qRT-PCR was conducted with MonAmp™ Taqman qPCR Mix (MQ30101S, MQ30101S, Monad, China) on a fluorescence quantitative PCR instrument (CFX96, Bio-rad, USA). The relative expression level of targeted genes was analyzed using 2–ΔΔCt method and β-actin was regarded as the control gene. All sequences of primers are provided in Table 1 .
4
mRNA Extraction and RT-qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mRNA was extracted using Beyozol reagent (Beyotime). The concentration and purity of the mRNA were assessed by a Nanodrop spectrophotometer. The isolated mRNA (2 μg) was used for reverse transcription PCR to produce cDNA with an iScript cDNA synthesis kit (Bio-Rad). A total of 2 μL of cDNA product was used for subsequent RT–qPCR analysis using SYBR1 Premix Ex Taq (Takara, Dalian, Japan). All primers used in this study are shown in Supplementary file 3 .
5
Extraction and qPCR Analysis of mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mRNA was extracted using Beyozol reagent (Beyotime, Shanghai, China). The concentration and purity of mRNA were assessed by NanoDrop-2000 (Thermo Fisher, United States). PrimeScript RT Master Mix (Takara, Dalian, Japan) was used for reverse transcription. For PCR amplification, a total of 2 μl cDNA product was used for subsequent RT-qPCR analysis using SYBR1 Premix Ex TaqTM (Takara). The reaction system includes 10 μl of Forget-Me-Not qPCR Master Mix (Biotium, United States), 1 μl primer, and 7 μl RNase Free H2O as well. CFX96 Touch Real-Time PCR Detection System (Bio-Rad, United States) was utilized for reaction process. The following are primer sequences for ALP, OCN, and Osterix (Supplementary Table S1 ).
6
RNA Extraction and RT-qPCR Analysis in Yeast and HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from yeast cell or HeLa cell with RNAiso Plus (TaKaRa Bio, China) or Beyozol reagent (Beyotime) respectively. For yeast cell, cells (OD600nm of 5) were pretreated with 50 U lyticase at 30 °C for 30 min to increase extraction efficiency. Reverse transcription reactions were conducted using a PrimeScript RT reagent kit with gDNA eraser (Takara Bio, China). The primers for real-time quantitative PCR (RT-qPCR) are indicated in Table 3 . RT-qPCR experiments were performed using SYBR Premix Ex Taq II (TaKaRa Bio, China) and Bio-Rad CFX manager RT-qPCR system. Data were collected and analyzed by Bio-Rad CFX manager software.
7
Assessing Gene Expression in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment with BSHX decoction, day 2 adult nematodes were collected in a microfuge tube, and total RNA was isolated with Beyozol reagent (Beyotime Institute of Biotechnology) and reverse-transcribed into cDNA by using PrimeScript™ RT Master Mix (Takara Bio, Inc., Shiga, Japan). Transcript levels of the genes of interest were normalized to that of act-3 as the control. The cDNA products were amplified using qRT-PCR with LightCycler® RNA Master SYBR Green I (Roche Life Science, Penzberg, Germany), and the primers are listed in Supplementary Table S1 .
8
C. elegans Transcriptional Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Age-synchronized embryos were maintained on NGM plates seeded with bacteria at 16°C for 36 h and then at 23°C for 36 h. Total RNA was isolated with Beyozol reagent (Beyotime Institute of Biotechnology) and reverse-transcribed into complementary DNA using PrimeScript™ RT Master Mix (Takara Bio, Inc., Shiga, Japan). Transcript levels of the genes of interest were normalized to that of act-3 as a control. The cDNA products were amplified by qRT-PCR using LightCycler® RNA Master SYBR Green I (Roche Life Science, Penzberg, Germany) and the primers listed in Supplementary Table S2 .
9
Quantifying Circular RNA Stability with 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 tissue samples and cell lysates using the Beyozol reagent (Beyotime, Shanghai, China). BeyoRT™ III First Strand cDNA Synthesis Kit (Beyotime) was used for the reverse transcription assay. BeyoFast™ SYBR Green qPCR Mix Kit (Beyotime) was used for PCR detection. The relative expression level of each gene was calculated using the 2-∆∆Ct technique.17 Glyceraldehyde-phosphate dehydrogenase (GAPDH) for circ_0010235 or HOXA10 and U6 for miR-588 served as the internal controls. For analysis of stability, circ_0010235 and ALDH4A1 levels were determined by RT-qPCR after total RNA was performed with the treatment of RNase R (GENESEED). Reverse transcription using random or oligo (dT)18 primers were used to identify the circular structure, followed by expression quantification of circ_0010235 and ALDH4A1. Table 2 shows the primer sequences.
10
Mulberry Leaf RNA Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We harvested fresh and young mulberry leaves from the Mulberry Garden of Jiangsu University, Zhenjiang, China and were identified as leaves of Morus alba L. by Professor Zhen Ouyang. Three well-grown mulberry trees were selected and the first to third tender leaf at the top were collected every ten days from July 15 to November 15, 2022 at 9:00 a.m. These leaves were quickly frozen with liquid nitrogen, and stored at -80 °C. To extract RNA, we used Beyozol reagent (Beyotime Biotechnology, Shanghai, China), and subsequently converted it into cDNA using a first-strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was stored at -20 °C for later use.
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!