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:1/18/2017)... ... January 18, 2017 , ... uBiome, the leading microbial genomics ... Science Editor, Dr. Elisabeth Bik, in the December 2016 issue of the Dutch ... in October 2016 from her previous position at Stanford University School of Medicine ...
(Date:1/18/2017)...  Caris Life Sciences, a leading innovator in ... private funder of pancreatic cancer research, are collaborating ... of immunotherapy in the treatment of pancreatic cancer. ... identify potential trial candidates based on biomarker expression ... study investigators. The Lustgarten Foundation is a sponsor ...
(Date:1/17/2017)... West Point, IA (PRWEB) , ... January 17, ... ... approval from China for Balance™ GT soybeans. The new Balance™ GT Soybean Performance ... tolerance to glyphosate and isoxaflutole, the active ingredient in the new Balance® Bean ...
(Date:1/17/2017)... Ind. , Jan. 17, 2017  Zimmer Biomet ... fourth quarter and full-year 2016 sales and earnings conference ... Tuesday, January 31, 2017, at 8 a.m. Eastern Time.  ... will be made available at 7:30 a.m. Eastern Time ... live audio webcast can be accessed via Zimmer Biomet,s ...
Breaking Biology Technology:
(Date:12/20/2016)... and GENEVA, Dec, 20, 2016   Valencell , ... technology, and STMicroelectronics (NYSE: STM), a global ... electronics applications, announced today the launch of a ... biometric wearables that includes ST,s compact SensorTile ... Benchmark™ biometric sensor system. Together, SensorTile and ...
(Date:12/16/2016)... , Dec. 16, 2016 The global wearable medical ... 12.14 billion by 2021 from USD 5.31 billion in 2016, at ... ... mainly driven by technological advancements in medical devices, launch of a ... preference for wireless connectivity among healthcare providers, and increasing focus on ...
(Date:12/15/2016)... 2016 Advancements in biometrics will ... and wellbeing (HWW), and security of vehicles ... passenger vehicles begin to feature fingerprint recognition, ... beat monitoring, brain wave monitoring, stress detection, ... pulse detection. These will be driven by ...
Breaking Biology News(10 mins):