Robust RNA Extraction and Sequencing from ACL Tissues

RNA extracted from ACL tissues was pulverized into a powder using a dismembranator (Mikro-S, Sartorius, Melsungen, Germany) under liquid nitrogen. Total RNA was extracted using the miRNeasy kit (Qiagen, Manchester, United Kingdom) according to the manufacturer’s instructions (Peffers et al., 2020 (link)). The RNA samples were quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, United States). The integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer (Agilent, Stockport, United Kingdom) using an RNA Pico chip (Agilent, Stockport, United Kingdom). Then, 1000 ng RNA per ACL sample was submitted for library preparation using NEBNext^® Small RNA Library Prep Set for Illumina (New England Biosciences (NEB), Ipswich, United States) but with the addition of a Cap-Clip™ Acid Pyrophosphatase (Cell script, Madison, United States) step to remove any 5′ cap structures on some snoRNAs (Steinbusch et al., 2017 (link)) and size selected using a range 120–300 bp (including adapters). These steps enabled both miRNAs and snoRNAs to be identified using an unbiased approach. The pooled libraries were sequenced on an Illumina HiSeq 4000 platform with sequencing chemistry version 1 to generate 2 × 150 bp paired-end reads.

Free full text: Click here

Kharaz Y.A., Zamboulis D.E., Fang Y., Welting T.J., Peffers M.J, & Comerford E.J. (2023). Small RNA signatures of the anterior cruciate ligament from patients with knee joint osteoarthritis. Frontiers in Molecular Biosciences, 10, 1266088.

Publication 2023

Mikro-S miRNeasy kit NanoDrop spectrophotometer Agilent 2100 Bioanalyzer RNA Pico chip Cap-Clip™ Acid Pyrophosphatase HiSeq 4000

Corresponding Organization : University of Liverpool

Other organizations : Royal Veterinary College, Maastricht University Medical Centre

Top 5 similar protocols

Variable analysis

independent variables

Pulverization of ACL tissues using a dismembranator under liquid nitrogen

dependent variables

Identification of miRNAs and snoRNAs in ACL samples

control variables

Quantity of RNA used for library preparation (1000 ng per ACL sample)
Size selection of RNA fragments (120-300 bp including adapters)

controls

Positive control: None mentioned
Negative control: None 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!