Laser capture microdissection (LCM) and whole genome amplification (WGA) of DNA from normal breast tissue - optimization for genome wide array analyses.
Aaltonen, Kristina LU ; Ebbesson, Anna LU ; Wigerup, Caroline LU and Hedenfalk, Ingrid LU
(2011)
In BMC Research Notes
4.
p.69-69
- Abstract
- Background
Laser capture microdissection (LCM) can be applied to tissues where cells of interest are distinguishable from surrounding cell populations. Here, we have optimized LCM for fresh frozen normal breast tissue where large amounts of fat can cause problems during microdissection. Since the amount of DNA needed for genome wide analyses, such as single nucleotide polymorphism (SNP) arrays, is often greater than what can be obtained from the dissected tissue, we have compared three different whole genome amplification (WGA) kits for amplification of DNA from LCM material. In addition, the genome wide profiling methods commonly used today require extremely high DNA quality compared to PCR based techniques and DNA quality is... (More) - Background
Laser capture microdissection (LCM) can be applied to tissues where cells of interest are distinguishable from surrounding cell populations. Here, we have optimized LCM for fresh frozen normal breast tissue where large amounts of fat can cause problems during microdissection. Since the amount of DNA needed for genome wide analyses, such as single nucleotide polymorphism (SNP) arrays, is often greater than what can be obtained from the dissected tissue, we have compared three different whole genome amplification (WGA) kits for amplification of DNA from LCM material. In addition, the genome wide profiling methods commonly used today require extremely high DNA quality compared to PCR based techniques and DNA quality is thus critical for successful downstream analyses.
Findings
We found that by using FrameSlides without glass backing for LCM and treating the slides with acetone after staining, the problems caused by excessive fat could be significantly decreased. The amount of DNA obtained after extraction from LCM tissue was not sufficient for direct SNP array analysis in our material. However, the two WGA kits based on Phi29 polymerase technology (Repli-g® (Qiagen) and GenomiPhi (GE Healthcare)) gave relatively long amplification products, and amplified DNA from Repli-g® gave call rates in the subsequent SNP analysis close to those from non-amplified DNA. Furthermore, the quality of the input DNA for WGA was found to be essential for successful SNP array results and initial DNA fragmentation problems could be reduced by switching from a regular halogen lamp to a VIS-LED lamp during LCM.
Conclusions
LCM must be optimized to work satisfactorily in difficult tissues. We describe a work flow for fresh frozen normal breast tissue where fat is inclined to cause problems if sample treatment is not adapted to this tissue. We also show that the Phi29-based Repli-g® WGA kit (Qiagen) is a feasible approach to amplify DNA of high quality prior to genome wide analyses such as SNP profiling. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/2298252
- author
- Aaltonen, Kristina
LU
; Ebbesson, Anna
LU
; Wigerup, Caroline
LU
and Hedenfalk, Ingrid
LU
- organization
- publishing date
- 2011
- type
- Contribution to journal
- publication status
- published
- subject
- in
- BMC Research Notes
- volume
- 4
- pages
- 69 - 69
- publisher
- BioMed Central (BMC)
- external identifiers
-
- pmid:21418556
- scopus:85040644927
- DOI
- 10.1186/1756-0500-4-69
- language
- English
- LU publication?
- yes
- id
- c32d8143-5590-4b30-9046-3526c9a801e7 (old id 2298252)
- alternative location
- http://www.biomedcentral.com/1756-0500/4/69
- http://www.ncbi.nlm.nih.gov/pubmed/21418556
- date added to LUP
- 2016-04-04 14:38:12
- date last changed
- 2022-01-30 02:22:32
@article{c32d8143-5590-4b30-9046-3526c9a801e7,
abstract = {{Background<br/><br>
Laser capture microdissection (LCM) can be applied to tissues where cells of interest are distinguishable from surrounding cell populations. Here, we have optimized LCM for fresh frozen normal breast tissue where large amounts of fat can cause problems during microdissection. Since the amount of DNA needed for genome wide analyses, such as single nucleotide polymorphism (SNP) arrays, is often greater than what can be obtained from the dissected tissue, we have compared three different whole genome amplification (WGA) kits for amplification of DNA from LCM material. In addition, the genome wide profiling methods commonly used today require extremely high DNA quality compared to PCR based techniques and DNA quality is thus critical for successful downstream analyses.<br/><br>
Findings<br/><br>
We found that by using FrameSlides without glass backing for LCM and treating the slides with acetone after staining, the problems caused by excessive fat could be significantly decreased. The amount of DNA obtained after extraction from LCM tissue was not sufficient for direct SNP array analysis in our material. However, the two WGA kits based on Phi29 polymerase technology (Repli-g® (Qiagen) and GenomiPhi (GE Healthcare)) gave relatively long amplification products, and amplified DNA from Repli-g® gave call rates in the subsequent SNP analysis close to those from non-amplified DNA. Furthermore, the quality of the input DNA for WGA was found to be essential for successful SNP array results and initial DNA fragmentation problems could be reduced by switching from a regular halogen lamp to a VIS-LED lamp during LCM.<br/><br>
Conclusions<br/><br>
LCM must be optimized to work satisfactorily in difficult tissues. We describe a work flow for fresh frozen normal breast tissue where fat is inclined to cause problems if sample treatment is not adapted to this tissue. We also show that the Phi29-based Repli-g® WGA kit (Qiagen) is a feasible approach to amplify DNA of high quality prior to genome wide analyses such as SNP profiling.}},
author = {{Aaltonen, Kristina and Ebbesson, Anna and Wigerup, Caroline and Hedenfalk, Ingrid}},
language = {{eng}},
pages = {{69--69}},
publisher = {{BioMed Central (BMC)}},
series = {{BMC Research Notes}},
title = {{Laser capture microdissection (LCM) and whole genome amplification (WGA) of DNA from normal breast tissue - optimization for genome wide array analyses.}},
url = {{http://dx.doi.org/10.1186/1756-0500-4-69}},
doi = {{10.1186/1756-0500-4-69}},
volume = {{4}},
year = {{2011}},
}