The total RNA for all samples was isolated using the RNeasy Plant Mini Kit (Qiagen) and any contaminating genomic DNA was degraded with TURBO DNase kit (Ambion) according to the manufacturer's instructions. RNA was diluted to 10 ng/μL and first-strand cDNA and qPCR were performed using Luna Universal One-Step RT-qPCR master mix (New England Biolabs) in a 20-µL reaction. qPCR analysis was performed on a 7500 Real Time PCR System (Applied Biosystems) using the UBQ10 (At4G05320) gene from A. thaliana as an internal reference (29 (link)). qPCR was performed in a 96-well optical PCR plate (ABgene) using the following parameters: 1 cycle of 10 min at 95 °C,and 40 cycles of 15 s at 95 °C, 15 s at 58 °C and 15 s at 65 °C, and 1 cycle of dissociation from 58 to 95 °C with 1 ° temperature increments. The qPCR primers used in this study are presented in SI Appendix, Table S1 .
Protocol detail
RT-qPCR Analysis of Plant Transcripts
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
RNeasy Plant Mini Kit
TURBO DNase kit
Luna Universal One-Step RT-qPCR master mix
7500 Real Time PCR System
Cdna
Dnase
Gene
Genomic
Plant
Primers
Corresponding Organization :
Other organizations : University of Copenhagen, University of Wisconsin–Madison, Saitama University
Top 5 similar protocols
Variable analysis
independent variables
- None explicitly mentioned
- Gene expression levels
- Total RNA isolated using RNeasy Plant Mini Kit (Qiagen)
- Contaminating genomic DNA degraded with TURBO DNase kit (Ambion)
- RNA diluted to 10 ng/μL
- First-strand cDNA and qPCR performed using Luna Universal One-Step RT-qPCR master mix (New England Biolabs)
- QPCR analysis performed on a 7500 Real Time PCR System (Applied Biosystems)
- UBQ10 (At4G05320) gene from A. thaliana used as internal reference
- QPCR parameters: 1 cycle of 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 15 s at 58 °C and 15 s at 65 °C, and 1 cycle of dissociation from 58 to 95 °C with 1 ° temperature increments
- Positive control: UBQ10 (At4G05320) gene from A. thaliana used as internal reference
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!