Extracting RNA from symbiotic fungi, bacteria and yeast in plant nodules.
Hi Dear Emily
To response to your question
I recommend you the paper that you find in attachment.
wish you luck
'Reverse transcription-PCR (RT-PCR) of total root and root hair RNA
RNA was isolated using the VS total RNA isolation kit (Promega, Madison, WI). For extraction of root hair RNA, the 50 ml tube (see above) was rinsed with 100 ml of the VS kit extraction buffer to collect the root hairs. For extraction of total root RNA, four 10 mm long lateral roots (including the root tips), grown as described above were used. A fraction of the extracted root hair RNA (50%) and total root RNA (5%) was used for cDNA reverse transcription using Moloney murine leukemia virus reverse transcriptase (Life Technologies) with a poly-A+ primer. The amounts of cDNA to be
used as templates were standardized with ubiquitin. Primers (forward: TTGAAGGAAACGGTGG; reverse: GTAACCTTCAAGAGCCTTG) were designed based on homologous regions of MtPR10-1 (Gamas et al., 1998; accession number Y08641) and PsRH2 (Mylona et al., 1995; accession number S74512). PCR conditions were an initial denaturation step of 5 min at 95 °C; 12±20 cycles of 45 s at 95 °C, 45 s at 54 °C and 45 s at 72 °C; and a ®nal elongation step of 10 min at 72 °C. PCR products were separated by electrophoresis, transferred to nylon membranes at alkaline pH and hybridized with a 32P-labelled probe generated by PCR using the same primers. Autoradiographs were analysed with a hosphorimager (Molecular Dynamics, Sunnyvale, CA).'
Source:
RESEARCH PAPER
A method for the isolation of root hairs from the model legume Medicago truncatula
Javier Ramos and Ton Bisseling
Journal of Experimental Botany, Vol. 54, No. 391, pp. 2245±2250, October 2003
Dear Emiliy,
my suggestion for RNA extraction is TRIzol protocol, that will allow you to exclude all DNA and proteic material. Here you can find very useful insights: https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf
Good luck with your work,
Davide
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