Venom Gland RNA Extraction and Purification

The venom gland tissue was homogenized in a 1 ml glass homogenizer with TRIzol solution (Invitrogen, Calsbad, CA, USA) aseptically. Then, 20% chloroform was added, and the sample was centrifuged and treated with RNA-free DNAase I to separate RNA from the cellular debris and residual DNA. The separated RNA was then pelleted using isopropyl alcohol and washed with 75% ethanol. The polyadenylated mRNA (poly(A)⁺ mRNA) was purified with oligo (dT) magnetic beads from 20 μg of total RNA, as per manufacturer’s instructions (Illumina, San Diego, CA, USA). The quality of the purified RNA was assessed immediately using the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). The RNA integrity number (RIN) of the sample was determined to be 8.6, indicating that the RNA was in good condition for downstream transcriptomic analysis.

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Chong H.P., Tan K.Y., Tan N.H, & Tan C.H. (2019). Exploring the Diversity and Novelty of Toxin Genes in Naja sumatrana, the Equatorial Spitting Cobra from Malaysia through De Novo Venom-Gland Transcriptomics. Toxins, 11(2), 104.

Publication 2019

TRIzol solution oligo (dT) magnetic beads Agilent 2100 Bioanalyzer

Cellular Chloroform Dnaase Ethanol I rna Isopropyl alcohol Oligo Polyadenylated mrna Rna i Tissue Transcriptomic analysis Trizol Venom

Corresponding Organization : University of Malaya

Top 5 similar protocols

Variable analysis

independent variables

Homogenization of venom gland tissue in TRIzol solution
Addition of 20% chloroform
Treatment with RNA-free DNAase I
Purification of polyadenylated mRNA (poly(A)+ mRNA) using oligo (dT) magnetic beads

dependent variables

Separation of RNA from cellular debris and residual DNA
Quality of the purified RNA (assessed using Agilent 2100 Bioanalyzer)
RNA integrity number (RIN) of the sample

control variables

Volume of TRIzol solution used (1 ml)
Aseptic conditions during homogenization
Manufacturer's instructions followed for mRNA purification

positive controls

None specified

negative controls

None specified

Annotations

Based on most similar protocols

Use a 1 ml glass homogenizer to homogenize the venom gland tissue in TRIzol solution (Invitrogen, Carlsbad, CA, USA) under aseptic conditions (Input protocol, Protocol 1, Protocol 2, Protocol 3, Protocol 4, Protocol 5).

Add 20% chloroform to separate the RNA (aqueous state) from the DNA and proteins (interface and organic states) (Input protocol, Protocol 1, Protocol 2, Protocol 3, Protocol 4, Protocol 5).

Treat the sample with RNA-free DNAase I (Thermo Fisher Scientific, MA, USA) to remove residual DNA (Input protocol, Protocol 1, Protocol 2, Protocol 3, Protocol 4, Protocol 5).

Precipitate the RNA with isopropyl alcohol and wash with 75% ethanol (Input protocol, Protocol 1, Protocol 2, Protocol 3, Protocol 4, Protocol 5).

Purify the polyadenylated mRNA (poly(A)+ mRNA) from 20 μg of total RNA using oligo (dT) magnetic beads (Illumina, San Diego, CA, USA) (Input protocol, Protocol 2, Protocol 3, Protocol 4, Protocol 5).

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