RNA was extracted with the RNAsimple kit (Tiangen) from germ-tubes grown for 18 h in YEPD liquid medium. The cDNAs were amplified with the PrimeScript® RT reagent kit (TaKaRa). Quantitative PCR experiments were performed using an ABI 7500 real-time detection system (Applied Biosystems, USA) with an SYBR Green reaction mix containing 10 μL of 2 × SYBR green premix, 2 μL of template, 0.4 μL of forward primer (10 mM) and reverse primer (10 mM), and 7.2 mL of nucleotide-free water. The PCR program included an initial denaturation step at 95°C for 30 s and then 40 amplification cycles at 95.0°C for 5 s and 60°C for 34 s. To ensure specificity, only primers that generated a single peak in the melting curve were selected. Primers used for qRT-PCR analysis are listed in S1 Table and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference. Each experiment included two technology replicates and was repeated at least three times.
Rnasimple kit
Manufactured by Tiangen Biotech
Sourced in China
The RNAsimple kit is a product designed for the extraction and purification of RNA from a variety of biological samples. It provides a simple and efficient method for isolating high-quality RNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Fastking rt kit, by Tiangen Biotech (9 mentions)
Gotaq qpcr master mix, by Promega (8 mentions)
Transzol, by Transgene (8 mentions)
Revertaid first strand synthesis kit, by Thermo Fisher Scientific (7 mentions)
Superreal premix color, by Tiangen Biotech (5 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Abi 7500 real time detection system, by Thermo Fisher Scientific (2 mentions)
Tb green premix ex taq 2, by Takara Bio (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Fastfire qpcr premix, by Tiangen Biotech (2 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Rnase r, by Illumina (2 mentions)
Rneasy minelute cleaning kit, by Qiagen (2 mentions)
Fastking one step rt pcr kit, by Tiangen Biotech (1 mentions)
Hiscript 2 q rt supermix for qpcr, by Vazyme (1 mentions)
Microrna rt kit, by Thermo Fisher Scientific (1 mentions)
Superreal premix color kit, by Tiangen Biotech (1 mentions)
Trizol, by Takara Bio (1 mentions)
Mix x mirna synthesis kit, by Takara Bio (1 mentions)
41 protocols using rnasimple kit
1
Quantitative PCR Analysis of Gene Expression
2
Quantitative RT-PCR Analysis of Wnt Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from tissues and from cells 48 h post-transfection using an RNA Simple kit (Tiangen Biotech, Co., Ltd.), and the RNA concentration and purity were determined using a micro nucleic acid quantitative analyzer. Subsequently, RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent kit (cat. no. RR036Q; Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions, and cDNA was amplified via qPCR using TB Green Premix Ex Taq II (cat. no. RR420A; Takara Biotechnology Co., Ltd.) as follows: 95°C for 5 min; followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the sequences were as follows: TCF3, forward 5′-AGGAGAAGGAGGACGAGGAG-3′ and reverse, 5′-AAAGGCCTCGTTGATGTCAC-3′; Wnt1 forward, 5′-CGGCGTTTATCTTCGCTATC-3′ and reverse, 5′-GCCTCGTTGTGAAGGTT-3′; β-catenin forward, 5′-GAAACGGCTTTCAGTTGAGC-3′ and reverse, 5′-CTGGCCATATCCACCAGAGT-3′; c-myc forward, 5′-AAAGGCCCCCAAGGTAGTTA-3′ and reverse, 5′-GCACAAGAGTTCCGTAGCTG-3′; and β-actin forward, 5′-GCTCTTTTCCAGCCTTCCTT-3′ and reverse, 5′-GAGCCAGAGCAGTGATCTCC-3′. β-actin served as the internal control for RT-qPCR. Relative expression levels were calculated using the 2−ΔΔCq method (17 (link)).
3
qPCR Transcriptomic Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal amounts of experimental cells were subjected to isolation of total RNAs by using the RNA simple Kit (Tiangen Biotech Co., Beijing, China). Then, 500 ng of total RNAs was added in a reverse-transcriptase reaction to generate the first strand of cDNA (with the Revert Aid First Strand Synthesis Kit from Thermo, Waltham, MA, USA). The synthesized cDNA fragments were served as the template for qPCR, in the GoTaq®qPCR Master Mix (from Promega), before being deactivated at 95°C for 10 min, and then amplified by 40 reaction cycles of the annealing at 95°C for 15 s and then extending at 60°C for 30 s. The final melting curve was validated to examine the amplification quality, whereas the mRNA expression level of β-actin served here as an optimal internal standard control. All the primers used for qPCR (Table S1 ) were synthesized by Tsingke (Chengdu, China).
4
RNA Extraction and qRT-PCR Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to the manufacturer's instructions, the total cellular RNA was extracted using RNAsimple kit (TianGen, Beijing, China), and the RNA reverse transcription was prepared using the FastKing RT Kit (TianGen). qRT-PCR was performed on a 7500 Real-Time PCR System (ABI, Foster City, CA, USA) using SYBR reagent (TianGen) following the manufacturer's instructions. The relative expression data of each gene was calculated using 2−ΔΔCT method, with ACTB as the internal reference gene. And the sequences of primer are shown in Table S1 .
5
Quantitative Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primers were synthesized by BGI (China), and the sequences are listed in Table 1 . Total RNA was isolated from MDA-MB-231 using the RNA simple kit (Tiangen, China) following the manufacturer's instructions. cDNA was synthesized using the HiScript II Q RT SuperMix for qPCR (Vazyme, China), and quantitative real-time PCR (Q-RT PCR) was performed using Fastking One Step RT-PCR kit (Tiangen, China). GAPDH was used as the reference gene. The ∆∆Ct method was used to analyze the real-time PCR data.
6
Quantification of Cellular RNA Species
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TransZol (TransGen) was used to lyse tissues and cells. RNA was extracted from the lysates with an RNAsimple kit (Tiangen). cDNA was synthesized according to the instruction of FastKing RT Kit (Tiangen) and MicroRNA RT Kit (Thermo Fisher). Then, SuperReal PreMix Color Kit (Tiangen) was utilized to detect the amount of circRNA/miRNA/mRNA as per the guidebook. The quantitative real‐time polymerase chain reaction (qRT‐PCR) was cycled at 95°C for 15 min, 40 cycles at 95°C for 10 s, and 60°C for 20 s. Obtained data were assessed using the 2−∆∆Ct method. U6 and GAPDH were used as internal controls. The primer sequences are displayed in Table S2 .
7
Quantitative Analysis of circRNA, miRNA, and mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues, cells and exosomes were lysed using TRIzol (TaKaRa, Dalian, China), and RNA was isolated with an RNAsimple kit (Tiangen, Beijing, China). After that, cDNA was synthesized with a primeScript™ synthesis kit (TaKaRa) or MiX-x™ miRNA synthesis Kit (TaKaRa). For the detection of the expression levels of circRNA/miRNA/mRNA, SYBR® Premix DimerEraser Kit (TaKaRa) was employed. Data were assessed by the 2−∆∆Ct method with U6 and β-actin as references. The sequences of sense and antisense primers were circ-RNF121 5′-CTCATCGCAACCTTGGTG-3′ and 5′-GACCCTCCATTGCTCTTCT-3′, RNF121 5′-ACCGTGGCCATGAAGCTATG-3′and 5′-GGTCACCATATTGTAGGAGCGT-3′, miR-1224-5p 5′-ACACTCCAGCTGGGGTGAGGACTCGGG-3′ and 5′-TGGTGTCGTGGAGTCG-3′, FOXM1 5′-CTTCTGGACCATTCACCC-3′ and 5′-CTCTGGATTCGGTCGTTT-3′, U6 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′, β-actin 5′-CACCATTGGCAATGAGCGGTTC-3′ and 5′-AGGTCTTTGCGGATGTCCACGT-3′.
8
Quantifying circRNA and miRNA Expressions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CRC tissues and cells were lysed with TransZol (TransGen, Beijing, China), and total RNA was extracted using an RNAsimple kit (Tiangen, Beijing, China). After performing concentration measure, RNA was reversely transcribed into cDNA using a FastKing RT Kit (Tiangen) or MiX-x™ synthesis Kit (TaKaRa, Dalian, China). Following that, SuperReal PreMix Color (Tiangen) was performed to detect the expression levels of circASXL1, miR-1205, or GRIK3. Data were assessed with the 2-∆∆Ct method with U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as references. The sequences of forward (F) and reverse (R) primers were listed in Table S1 .
9
Western Blot and qRT-PCR Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After washing twice using PBS, cells were lysed in RIPA Lysis Buffer (P0013B, Beyotime) including protease and phosphatase inhibitor cocktail. The lysates were then eluted in SDS loading buffer. After denaturation, the lysates were separated with SDS-PAGE. The proteins were then transferred to nitrocellulose membranes (10600001, GE Healthcare). After that, the membranes were blocked using QuickBlock™ Blocking Buffer (P0252, Beyotime) for 15 min at room temperature. Next, the membranes were interacting with the primary antibody at room temperature for 2 h. After washing for 30 min using TBS, the membranes were bound with the secondary antibody. After washing, the proteins were detected using enhanced chemiluminescence (ECL; P0018FS, Beyotime). The ImageJ software (NIH, USA) was used to analyze the target protein bands.
Total RNA extraction was performed using RNAsimple Kit (DP419, TIANGEN) according to the protocols. The RNA was reverse-transcribed using PrimeScript RT Master Mix (Takara, Japan). Quantitative real-time PCR analysis was performed using
BeyoFast™ SYBR Green qPCR Mix (2X) (D7260, Beyotime) with cDNA and primers. Primer sequences were shown in Table S1 and Table S2 as previously reported (Lian et al., 2018b (link); Zhang et al., 2020 (link)). The RNA levels of samples were analyzed using LightCycler 96 system (Roche, Switzerland).
Total RNA extraction was performed using RNAsimple Kit (DP419, TIANGEN) according to the protocols. The RNA was reverse-transcribed using PrimeScript RT Master Mix (Takara, Japan). Quantitative real-time PCR analysis was performed using
BeyoFast™ SYBR Green qPCR Mix (2X) (D7260, Beyotime) with cDNA and primers. Primer sequences were shown in Table S1 and Table S2 as previously reported (Lian et al., 2018b (link); Zhang et al., 2020 (link)). The RNA levels of samples were analyzed using LightCycler 96 system (Roche, Switzerland).
10
Quantitative RT-PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TRIzol Reagent (Invitrogen, United States; 15596-026) (1 ml per well) was used to lyse cells, then the RNAs were extracted using RNAsimple Kit (TIANGEN, China; DP419). The concentration of RNAs was tested by DS-11 spectrophotometer (DeNovix, United States). RNA samples (2 μg) were mixed with PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Tokyo, Japan; RR047A) and then applied to the ProFlex™ PCR system (ABI, United States) to obtain cDNAs. The cDNA samples were mixed with SYBR Premix Ex Taq II (TaKaRa, Tokyo, Japan; RR820A) and quantified by using ABI QuantStudio 5 (ABI, United States); the reaction was initiated at 95°C for 1 min, then at 95°C for 5 s, and 60°C 30 s for a total of 40 cycles. The primers are listed in Supplementary Table S1 . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was set as the internal reference. The 2−ΔΔCt method was used to process the data.
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!