Navigation Links
RNA from LCM Samples

Juanita Gonzales, Ambion, Inc.


Obtaining specific populations of cells from heterogenous samples is a major challenge in studying gene expression profiles of specific cell types. Laser capture microdissection (LCM), developed in a collaborative effort between the NIH and Arcturus, Inc. specifically to isolate cancer cells from normal tissue, can be used to separate individual cell types from within complex tissues. RNA can then be extracted from these microdissected cells to analyze their gene expression.


How LCM Works
LCM can capture cells or discrete morphological structures from thin tissue sections. Frozen, O.C.T.-embedded tissue sections are prepared for the LCM process by fixation, staining, and dehydration. Sections are visualized through a thermoplastic film attached to the bottom of a microfuge tube cap. A laser pulse is directed through the film onto the target cells. The plastic film melts onto the targeted area, then cools and bonds with the underlying cells. The film, along with the adhered target cells, is collected (Figure 1). RNA can be isolated from these captured cells for real-time PCR and mRNA expression profiling. (See our tips article Getting Intact RNA from LCM Samples.)


Capture and Analyses of Dentate Gyrus from Mouse Brain
LCM was used to separate dentate gyrus from mouse brain sections (Figure 1). RNA from the captured dentate gyrus was purified using the RNAqueous-Micro Kit (see sidebar) and the purified RNA was assessed on an Agilent bioanalyzer (Figure 2). This RNA was of sufficient quality and amount for real-time RT-PCR analysis, which showed consistency between replicates and very low DNA contamination in the RNA samples purified with RNAqueous-Micro Kit (Figure 3).

Figure 1. LCM Collection of Dentate Gyrus from Mouse Brain. (A) Cryosection (10 micron) stained with cresyl violet. (B) Same section after Laser Capture Microdissection using PixCell IIe LCM workstation (Arcturus). Note absence of target structure (dentate gyrus). (C) Microdissected dentate gyrus adhered to LCM cap. The thermoplastic film with captured cells can be added directly to the RNAqueous-Micro lysis solution from the RNA extraction.

Figure 2. Electropherogram of RNA from Microdissected Dentate Gyrus. RNA was extracted from microdissected tissue using Ambion's RNAqueous-Micro Kit and analyzed on an Agilent 2100 bioanalyzer. RNA concentration =18 ng/l, rRNA [28S/18S rRNA] = 0.89.

Figure 3. Real-time RT-PCR Using RNA From Microdissected Mouse Brain Dentate Gyrus. RT-PCR was performed in duplicate RNA from the microdissected dentate gyrus. The assay was run on an ABI PRISM 7000 Sequence Detection System with primers and probe for Mouse GAPDH. Amplification from left to right represents female 810 wk and male 26 wk dentate gyrus, and minus-RT controls.


ba ck to top


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Quantitation of the Protein MIA as a Marker for Chondrocytes in Research Samples
2. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
3. Preserve Samples for RNA Expression Microarrays RNAlater Around the World
4. Robust High Throughput RNA Isolation from Blood Samples
5. RNAlater Around the World: The Art of Processing Difficult Samples
6. Working With Laser Capture Microdissection Samples
7. Bio-Plex Cytokine Immunoassays and ELISA: Comparison of Two Methodologies in Testing Samples From Asthmatic and Healthy Children, Rev A
8. Analysis of Bacterial Assemblage Genetic Diversity in Environmental Samples Using the DCode System
9. Fast Multi-Residue Pesticide Analysis in Soil and Vegetable Samples
10. Determination of Polar Organophosphorus Pesticides in Aqueous Samples by Direct Injection Using HPLC/MS/MS
11. Maintaining High Sensitivity when Working with Small-Volume Samples
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... Prostate Cancer Foundation (PCF) is pleased to announce 24 new Young ... cancer. Members of the Class of 2016 were selected from a pool of ... More About the Class of 2016 PCF Young Investigators ... ... ...
(Date:6/23/2016)... ... June 23, 2016 , ... ... the launch of the Supplyframe Design Lab . Located in Pasadena, Calif., ... the future of how hardware projects are designed, built and brought to market. ...
(Date:6/23/2016)... , June 23, 2016 Apellis ... Phase 1 clinical trials of its complement C3 ... single and multiple ascending dose studies designed to ... (PD) of subcutaneous injection in healthy adult volunteers. ... (SC) either as a single dose (ranging from ...
(Date:6/23/2016)... NEW YORK , June 23, 2016 ... the trading session at 4,833.32, down 0.22%; the Dow Jones ... the S&P 500 closed at 2,085.45, down 0.17%. Stock-Callers.com has ... INFI ), Nektar Therapeutics (NASDAQ: NKTR ... BIND Therapeutics Inc. (NASDAQ: BIND ). Learn more ...
Breaking Biology Technology:
(Date:5/3/2016)... May 3, 2016  Neurotechnology, a provider of ... MegaMatcher Automated Biometric Identification System (ABIS) , a ... projects. MegaMatcher ABIS can process multiple complex biometric ... combination of fingerprint, face or iris biometrics. It ... and MegaMatcher Accelerator , which have ...
(Date:4/28/2016)... First quarter 2016:   , Revenues amounted ... quarter of 2015 The gross margin was 49% (27) ... the operating margin was 40% (-13) Earnings per share ... operations was SEK 249.9 M (21.2) , Outlook   ... M. The operating margin for 2016 is estimated to ...
(Date:4/26/2016)... 2016 Research and Markets has ... Market 2016-2020"  report to their offering.  , ,     ... The analysts forecast the global multimodal biometrics market ... the period 2016-2020.  Multimodal biometrics is ... as the healthcare, BFSI, transportation, automotive, and government ...
Breaking Biology News(10 mins):