Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage

André E S Simões, Diane M. Pereira, Joana D. Amaral, Ana F. Nunes, Sofia E. Gomes, Pedro M. Rodrigues, Adrian C. Lo, Rudi D'Hooge, Clifford J. Steer, Stephen N Thibodeau, Pedro M. Borralho, Cecília M P Rodrigues

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol® and TRIzol®LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.Results: TRIzol®LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol® was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer's disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol®-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at -80°C until used for protein isolation.Simple modifications to the TRIzol® manufacturer's protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol® manufacturer's protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol®-chloroform fractions stored for up to 2 years at -80°C.Conclusions: We provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol® and TRIzol®LS compared to the manufacturer's protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer's disease mice model, stored in TRIzol®-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol® manufacturer's protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

Original languageEnglish (US)
Article number181
JournalBMC Genomics
Volume14
Issue number1
DOIs
StatePublished - Mar 15 2013

Fingerprint

RNA
Proteins
Colonic Neoplasms
Chloroform
trizol
Immunoblotting
Brain Neoplasms
Nucleic Acids
Alzheimer Disease
Colon
Staining and Labeling
Sonication
DNA Mismatch Repair
Selection Bias
DNA
Frontal Lobe
Post Translational Protein Processing
Nuclear Proteins
Adenoma
Urea

Keywords

  • Long-term sample storage in TRIzol®
  • Ponceau S as loading control in immunobloting
  • Protein extraction
  • Sonication
  • TRIzol® protein isolation

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Simões, A. E. S., Pereira, D. M., Amaral, J. D., Nunes, A. F., Gomes, S. E., Rodrigues, P. M., ... Rodrigues, C. M. P. (2013). Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage. BMC Genomics, 14(1), [181]. https://doi.org/10.1186/1471-2164-14-181

Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage. / Simões, André E S; Pereira, Diane M.; Amaral, Joana D.; Nunes, Ana F.; Gomes, Sofia E.; Rodrigues, Pedro M.; Lo, Adrian C.; D'Hooge, Rudi; Steer, Clifford J.; Thibodeau, Stephen N; Borralho, Pedro M.; Rodrigues, Cecília M P.

In: BMC Genomics, Vol. 14, No. 1, 181, 15.03.2013.

Research output: Contribution to journalArticle

Simões, AES, Pereira, DM, Amaral, JD, Nunes, AF, Gomes, SE, Rodrigues, PM, Lo, AC, D'Hooge, R, Steer, CJ, Thibodeau, SN, Borralho, PM & Rodrigues, CMP 2013, 'Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage', BMC Genomics, vol. 14, no. 1, 181. https://doi.org/10.1186/1471-2164-14-181
Simões, André E S ; Pereira, Diane M. ; Amaral, Joana D. ; Nunes, Ana F. ; Gomes, Sofia E. ; Rodrigues, Pedro M. ; Lo, Adrian C. ; D'Hooge, Rudi ; Steer, Clifford J. ; Thibodeau, Stephen N ; Borralho, Pedro M. ; Rodrigues, Cecília M P. / Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage. In: BMC Genomics. 2013 ; Vol. 14, No. 1.
@article{11d8cf8b1b8b4bd5bd62cf0bb274c676,
title = "Efficient recovery of proteins from multiple source samples after trizol{\circledR} or trizol{\circledR}LS RNA extraction and long-term storage",
abstract = "Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol{\circledR} and TRIzol{\circledR}LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.Results: TRIzol{\circledR}LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol{\circledR} was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer's disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol{\circledR}-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at -80°C until used for protein isolation.Simple modifications to the TRIzol{\circledR} manufacturer's protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol{\circledR} manufacturer's protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol{\circledR}-chloroform fractions stored for up to 2 years at -80°C.Conclusions: We provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol{\circledR} and TRIzol{\circledR}LS compared to the manufacturer's protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer's disease mice model, stored in TRIzol{\circledR}-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol{\circledR} manufacturer's protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.",
keywords = "Long-term sample storage in TRIzol{\circledR}, Ponceau S as loading control in immunobloting, Protein extraction, Sonication, TRIzol{\circledR} protein isolation",
author = "Sim{\~o}es, {Andr{\'e} E S} and Pereira, {Diane M.} and Amaral, {Joana D.} and Nunes, {Ana F.} and Gomes, {Sofia E.} and Rodrigues, {Pedro M.} and Lo, {Adrian C.} and Rudi D'Hooge and Steer, {Clifford J.} and Thibodeau, {Stephen N} and Borralho, {Pedro M.} and Rodrigues, {Cec{\'i}lia M P}",
year = "2013",
month = "3",
day = "15",
doi = "10.1186/1471-2164-14-181",
language = "English (US)",
volume = "14",
journal = "BMC Genomics",
issn = "1471-2164",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage

AU - Simões, André E S

AU - Pereira, Diane M.

AU - Amaral, Joana D.

AU - Nunes, Ana F.

AU - Gomes, Sofia E.

AU - Rodrigues, Pedro M.

AU - Lo, Adrian C.

AU - D'Hooge, Rudi

AU - Steer, Clifford J.

AU - Thibodeau, Stephen N

AU - Borralho, Pedro M.

AU - Rodrigues, Cecília M P

PY - 2013/3/15

Y1 - 2013/3/15

N2 - Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol® and TRIzol®LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.Results: TRIzol®LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol® was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer's disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol®-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at -80°C until used for protein isolation.Simple modifications to the TRIzol® manufacturer's protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol® manufacturer's protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol®-chloroform fractions stored for up to 2 years at -80°C.Conclusions: We provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol® and TRIzol®LS compared to the manufacturer's protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer's disease mice model, stored in TRIzol®-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol® manufacturer's protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

AB - Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol® and TRIzol®LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.Results: TRIzol®LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol® was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer's disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol®-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at -80°C until used for protein isolation.Simple modifications to the TRIzol® manufacturer's protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol® manufacturer's protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol®-chloroform fractions stored for up to 2 years at -80°C.Conclusions: We provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol® and TRIzol®LS compared to the manufacturer's protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer's disease mice model, stored in TRIzol®-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol® manufacturer's protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

KW - Long-term sample storage in TRIzol®

KW - Ponceau S as loading control in immunobloting

KW - Protein extraction

KW - Sonication

KW - TRIzol® protein isolation

UR - http://www.scopus.com/inward/record.url?scp=84874944258&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84874944258&partnerID=8YFLogxK

U2 - 10.1186/1471-2164-14-181

DO - 10.1186/1471-2164-14-181

M3 - Article

C2 - 23496794

AN - SCOPUS:84874944258

VL - 14

JO - BMC Genomics

JF - BMC Genomics

SN - 1471-2164

IS - 1

M1 - 181

ER -