Journal: Frontiers in Plant Science
Article Title: Laser Capture Microdissection Protocol for Xylem Tissues of Woody Plants
Figure Lengend Snippet: Suitability of RNA from LCM samples for gene expression analysis . (A) Aspen: reverse transcriptase-PCR of laser microdissected developing wood fibers of Populus tremula . RNA was amplified using the MessageAmp TM II aRNA kit (ThermoFischer) and then subjected to RT-PCR using actin and ubiquitin specific primers. PCR templates: 1 and 4, cDNA library constructed from Populus tremula x tremuloides developing wood total RNA; 2 and 5, cDNA library constructed from laser microdissected aspen fiber cells (starting from 4 ng of RNA); 3 and 6, cDNA library constructed from laser microdissected fiber cells (starting from 6 ng of RNA). PCR primers: 1 to 3, ACTIN ; 4 to 6, UBIQUITIN . L, 1 kb DNA ladder. PCR 35 cycles. (B) RNA sequencing, spruce: representative quality scores across all bases (Sanger/Illumina 1.9 encoding) for xylem tracheids collected by LCM. Y-axis – quality scores; the higher the score the better the base call. Green area – very good quality calls, orange – reasonable quality calls, red – calls of poor quality. Red line – median value, blue line – mean quality.
Article Snippet: Equipment and Reagents RNA extraction kit for LCM material/small samples (RNAqueous® Micro Kit, AMBION) (aspen); RNeasy® Plant Mini Kit (QIAGEN, 74904) (spruce); RNase free water (AMBION AM9930 (aspen); QIAGEN (spruce)); RNA assay kit (AGILENT RNA 6000 PICO KIT 5067-1513); Agilent 2100 G2938A Bioanalyzer for RNA quantitation and quality assessment.
Techniques: Laser Capture Microdissection, Expressing, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, cDNA Library Assay, Construct, RNA Sequencing Assay