Total RNA from the isolated RGCs was extracted using Trizol reagent (Invitrogen), purified using RNeasy mini-columns (QIAGEN), and treated with RNase-free DNase I (QIAGEN). The RNA purity was verified by confirming that the OD260 nm/OD280 nm ratio exceeded 1.9. cDNA was synthesized using the SuperScript II first-strand RT-PCR kit (Invitrogen). The gene expression of Polg, Ogg1, Myh, ND4, ND5, ND6, and CytB was measured by SYBR Green-based real-time PCR (Applied Biosystems, 7500 Fast). The primers for each gene of interest were designed for each target mRNA and are shown in Suppl Table 2. The β-actin gene was used as a normalization control. All qRT-PCR reactions used 40 ng of cDNA. The reactions were performed in duplicate in 2 separate experiments, and the transcripts were quantified using the ΔΔCt method.
Superscript 2 first strand rt pcr kit
Manufactured by Thermo Fisher Scientific
The SuperScript II first-strand RT-PCR kit is a laboratory tool used for reverse transcription and subsequent polymerase chain reaction (RT-PCR) to amplify specific RNA sequences. The kit provides the necessary components, including reverse transcriptase enzyme, dNTPs, and buffers, to facilitate the conversion of RNA into complementary DNA (cDNA) and the subsequent amplification of the cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by Qiagen (1 mentions)
Rneasy mini column, by Qiagen (1 mentions)
Plant rna reagent, by Thermo Fisher Scientific (1 mentions)
High sensitivity ngs fragment analysis kit, by Agilent Technologies (1 mentions)
Aati fragment analyzer, by Agilent Technologies (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
3 protocols using superscript 2 first strand rt pcr kit
1
Mitochondrial Gene Expression in Retinal Ganglion Cells
2
Quantification and Analysis of Spliced bZIP60 in Arabidopsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Arabidopsis seedlings or different organs were harvested in liquid nitrogen. RNA was extracted from seedlings and organs using TRIzol reagent (Invitrogen, Life Technologies) or Plant RNA Reagent (Invitrogen, Life Technologies) respectively. Subsequently total RNA was quantified using a Qubit fluorometer (Invitrogen, Life Technologies) and analyzed by CE-LIF (AATI Fragment Analyzer, Advanced Analytical Technologies) using STD SENSITIVITY TOTAL RNA ANALYSIS KIT (Advanced Analytical Technologies) to evaluate the RQN (RNA Quality Number). Total RNA was treated with DNase I (Fermentas, Thermo Scientific) and cDNA was synthesized using a SuperScript II first-strand RT-PCR kit (Invitrogen, Life Technologies). To amplify both the unspliced and spliced forms of bZIP60, the following primers were used: 5`-GCTGAAAACCAGTCTCTACGTT-3`and 5`-AAGCAGGGAACCCAACAG-3`. PCR conditions for amplification were: initial denaturation: 5 min at 95°C; 30 s at 95°C, 30 s at 58°C and 30 s at 72°C during 34 cycles. Subsequently, PCR products were resolved by gel electrophoresis on agarose (3.5% w/v) using TAE 1X as running buffer or prepared for CE-LIF using the High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies) and analyzed in the fragment analyzer instrument (AATI Fragment Analyzer, Advanced Analytical Technologies) following the manufacturer instructions.
3
Quantitative RT-PCR Analysis of Arabidopsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For quantitative RT-PCR analyses, the seedlings were grown for 1 week on 1/2 MS agar (0.7% w/v) and subsequently transferred for the indicated period of time into liquid 1/2 MS supplemented with indicated concentrations of estradiol and/or DTT. Total RNA was isolated using TRIzol reagent (Invitrogen, Life Technologies) according to manufacturer's protocol and treated with DNAse I (Fermentas, Thermo Scientific). For cDNA synthesis, the SuperScript II first-strand RT-PCR kit (Invitrogen, Life Technologies) was utilized. Arabidopsis UBIQUITIN (At5g25760) mRNA level was used as a control. Oligonucleotides used for PCR amplifications are listed in Supplemental 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!