PMID- 28817123
OWN - NLM
STAT- MEDLINE
DCOM- 20170830
LR  - 20201207
IS  - 1750-2799 (Electronic)
IS  - 1754-2189 (Print)
IS  - 1750-2799 (Linking)
VI  - 12
IP  - 9
DP  - 2017 Sep
TI  - Evaluation of telomere length in human cardiac tissues using cardiac quantitative 
      FISH.
PG  - 1855-1870
LID - 10.1038/nprot.2017.082 [doi]
AB  - Telomere length has been correlated with various diseases, including 
      cardiovascular disease and cancer. The use of currently available telomere-length 
      measurement techniques is often restricted by the requirement of a large amount 
      of cells (Southern-based techniques) or the lack of information on individual 
      cells or telomeres (PCR-based methods). Although several methods have been used 
      to measure telomere length in tissues as a whole, the assessment of 
      cell-type-specific telomere length provides valuable information on individual 
      cell types. The development of fluorescence in situ hybridization (FISH) 
      technologies enables the quantification of telomeres in individual chromosomes, 
      but the use of these methods is dependent on the availability of isolated cells, 
      which prevents their use with fixed archival samples. Here we describe an 
      optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that 
      bypasses the previous limitations by avoiding contributions from undesired cell 
      types. We have used this protocol on small paraffin-embedded cardiac-tissue 
      samples. This protocol describes step-by-step procedures for tissue preparation, 
      permeabilization, cardiac-tissue pretreatment and hybridization with a 
      Cy3-labeled telomeric repeat complementing (CCCTAA)(3) peptide nucleic acid (PNA) 
      probe coupled with cardiac-specific antibody staining. We also describe how to 
      quantify telomere length by means of the fluorescence intensity and area of each 
      telomere within individual nuclei. This protocol provides comparative 
      cell-type-specific telomere-length measurements in relatively small human cardiac 
      samples and offers an attractive technique to test hypotheses implicating 
      telomere length in various cardiac pathologies. The current protocol (from tissue 
      collection to image procurement) takes  approximately 28 h along with three overnight 
      incubations. We anticipate that the protocol could be easily adapted for use on 
      different tissue types.
FAU - Sharifi-Sanjani, Maryam
AU  - Sharifi-Sanjani M
AD  - Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, 
      Pennsylvania, USA.
FAU - Meeker, Alan K
AU  - Meeker AK
AD  - Departments of Pathology, Oncology and Urology, Johns Hopkins University School 
      of Medicine, Baltimore, Maryland, USA.
FAU - Mourkioti, Foteini
AU  - Mourkioti F
AUID- ORCID: 0000-0002-7119-6640
AD  - Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, 
      Pennsylvania, USA.
AD  - Cardiovascular Institute, Perelman School of Medicine, University of 
      Pennsylvania, Philadelphia, Pennsylvania, USA.
AD  - Department of Cell and Developmental Biology, Perelman School of Medicine, 
      University of Pennsylvania, Philadelphia, Pennsylvania, USA.
LA  - eng
GR  - P30 AR069619/AR/NIAMS NIH HHS/United States
PT  - Journal Article
DEP - 20170817
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 0 (Carbocyanines)
RN  - 0 (Peptide Nucleic Acids)
RN  - 0 (cyanine dye 3)
SB  - IM
MH  - Carbocyanines/chemistry
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Myocardium/*chemistry
MH  - Peptide Nucleic Acids/chemistry
MH  - Telomere/*chemistry
PMC - PMC7716275
MID - NIHMS1646107
EDAT- 2017/08/18 06:00
MHDA- 2017/08/31 06:00
PMCR- 2020/12/04
CRDT- 2017/08/18 06:00
PHST- 2017/08/18 06:00 [entrez]
PHST- 2017/08/18 06:00 [pubmed]
PHST- 2017/08/31 06:00 [medline]
PHST- 2020/12/04 00:00 [pmc-release]
AID - nprot.2017.082 [pii]
AID - 10.1038/nprot.2017.082 [doi]
PST - ppublish
SO  - Nat Protoc. 2017 Sep;12(9):1855-1870. doi: 10.1038/nprot.2017.082. Epub 2017 Aug 
      17.