Total RNA was extracted using a RNeasy microRNA isolation kit (Qiagen, Valencia, CA, U.S.) following the manufacturer's instructions. Samples were treated with DNase I, and then, Transcript-Uni Cell was used for cDNA synthesis. A quantitative PCR supermix was used for the assays (Transgene Biotech, Beijing, China). RNA concentrations were measured using a Nanodrop 2000 Spectrophotometer (Biolab, Scoresby, Victoria, Australia) at a wavelength of 260 nm. Samples for subsequent analyses were only used if their 260 : 280 nm absorbance ratios were >1.8. Quantitative- and reverse transcription-PCR assays were performed with an ABI 7500 real-time PCR instrument and a Fast 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, U.S.), respectively. Three replicates were conducted for all assays. The relative expression of genes was calculated by the comparative threshold cycle method as 2−ΔΔCt. The primers used for the amplification assays are shown in Table 2 .
Fast 96 well thermal cycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fast 96-well Thermal Cycler is a laboratory instrument designed for rapid DNA amplification. It features a 96-well format and can perform temperature cycling operations required for polymerase chain reaction (PCR) experiments.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy microrna isolation kit, by Qiagen (3 mentions)
Abi 7500 real time pcr instrument, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (1 mentions)
Step one plus system, by Transgene (1 mentions)
Power sybr green pcr master mix, by Transgene (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Qpcr supermix, by Transgene (1 mentions)
7500 real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
4 protocols using fast 96 well thermal cycler
1
Quantitative Analysis of Gene Expression
2
Quantitative Transcriptome Analysis of Oocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative reverse transcription PCR (qRT-PCR) was performed by using an Applied Biosystems Step One Plus System and Power SYBR Green PCR Master Mix (TransGen Biotech). RNA was extracted from 50 oocytes using QIAGEN RNeasy Mini Kit, and cDNA was generated by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using an ABI 7500 real-time PCR instrument and a Fast 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, United States). The sequences of all primers used are listed in Supplementary Table S1 . GAPDH was used as a reference gene. The relative expression of genes was calculated with the comparative threshold cycle (CT) method as 2−△△CT.
3
RNA Isolation and Gene Expression Analysis in GV Oocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 40 collected GV oocytes using a RNeasy micro-RNA isolation kit (Qiagen, Valencia, CA, and U.S.) following the manufacturer instructions. The RNA concentrations were measured using a NanoDrop 2000 Spectrophotometer (Biolab, Scoresby, Victoria, Australia) at wavelength of 260 nm. We wouldn't use the samples for subsequent analyses until their absorbance ratio at 260 nm: 280 nm >1.8.
Reverse transcription was conducted to generate cDNA libraries using a Quantitated Reverse Transcription Kit (Qiagen) according to the manufacturer instructions and we treated the sample with DNaseI before that. QRT-PCR and RT-PCR were performed using an ABI 7500 real-time PCR instrument and a Fast 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, U.S.). The sequences of all primers used are listed inSupplementary Table 1 . The relative expression of genes was calculated with the comparative threshold cycle (CT) method as 2−ΔΔCT.
Reverse transcription was conducted to generate cDNA libraries using a Quantitated Reverse Transcription Kit (Qiagen) according to the manufacturer instructions and we treated the sample with DNaseI before that. QRT-PCR and RT-PCR were performed using an ABI 7500 real-time PCR instrument and a Fast 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, U.S.). The sequences of all primers used are listed in
4
RNA Extraction and Analysis of Oocyte Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 40 GV, MI or MII oocytes using a RNeasy micro‐RNA isolation kit (Qiagen) following the manufacturer's instructions. Samples were treated with DNase I, and then Transcript‐Uni Cell was used for cDNA Synthesis. A Q‐PCR super‐mix was used for the assays (Trans Gen Biotech). RNA concentrations were measured using a Nanodrop 2000 Spectrophotometer (Biolab, Scoresby) at a wavelength of 260 nm. Samples for subsequent analyses were only used if their 260:280 nm absorbance ratios were >1.8. Primers for the published reference RNA sequences for real‐time Q‐PCR and RT‐PCR are listed in Table 1 .Q‐PCR and RT‐PCR assays were performed with an ABI 7500 real‐time PCR instrument and a Fast 96‐well Thermal Cycler (Applied Biosystems), respectively. Three replicates were conducted for all assays. The relative expression of genes was calculated with the comparative threshold cycle (CT) method as 2–ΔΔCT. The primers used for the amplification assays are shown in Table 2 .
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!