Laser Capture Microdissection for RNA-Seq

LCM-RNA-Seq was performed as described previously [9 (link), 60 (link)]. Briefly, Cryosections of OCT-embedded tissue blocks were transferred to PEN membrane glass slides and stained with cresyl violet acetate. Adjacent sections were H&E stained for pathology review. Laser capture microdissection was performed on a PALM MicroBeam microscope (Zeiss), collecting at least 1000 cells per compartment. RNA was extracted and libraries were prepared using the Ovation RNA-Seq System V2 kit (NuGEN). Libraries were sequenced to a depth of 30million, 100bp, single-end reads on an Illumina HiSeq 2000 platform. Reads and transcripts per million (TPM) were estimated for each transcript using the transcript sequences from the GENCODE Release 34 (GRCh38.p5) and the Salmon software (v1.3.0). Counts and TPM were summarized at the gene level by summing up the transcript values for each corresponding gene.

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.

Maurer H.C., Curiel-Garcia A., Holmstrom S., Laise P., Palermo C.F., Sastra S.A., Andren A., Li Z., LeLarge T., Sagalovskiy I., Ross D.R., Rosario V., Lu K., Ferraiuolo E., Spinosa N., Wong W., Shaw K., Chabot J.A., Genkinger J., Hibshoosh H., Manji G.A., Iuga A., Schmid R.M., Badgley M.A., Johnson K., Califano A., Lyssiotis C, & Olive K.P. (2023). Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer. bioRxiv.

Publication Preprint 2023

PALM MicroBeam microscope Ovation RNA-Seq System V2 kit HiSeq 2000 platform

Cells Cresyl violet acetate Cryosections Gene Laser capture microdissection Membrane Microscope Palm Rna seq Salmon Tissue

Corresponding Organization : Columbia University

Other organizations : Klinikum rechts der Isar, Technical University of Munich, The University of Texas Southwestern Medical Center, University of Michigan–Ann Arbor, Westfriesgasthuis, University of North Carolina at Chapel Hill, University of New Hampshire at Manchester, DarwinHealth (United States)

Top 5 similar protocols

Variable analysis

independent variables

Laser capture microdissection of tissue sections

dependent variables

Gene expression levels (transcripts per million, TPM)

control variables

Cryosections of OCT-embedded tissue blocks
Adjacent sections stained with H&E for pathology review
RNA extraction and library preparation using Ovation RNA-Seq System V2 kit (NuGEN)
Sequencing depth of 30 million, 100bp, single-end reads on Illumina HiSeq 2000 platform
Transcript quantification using Salmon software (v1.3.0) and GENCODE Release 34 (GRCh38.p5) transcript sequences

controls

No explicit positive or negative controls mentioned

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.

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?

Revolutionizing how scientists
search and build protocols!