Navigation Links
Isolate and Analyze Total RNA from Cells Harvested by Laser Capture ,,, Microdissection



StrataPrep total RNA microprep kit for very small samples

Karen Dolter Sylvia Norman Jeff Braman

Stratagene

Isolating total RNA from laser capture microdissection samples can be challenging due to the typically small size of the samples. The StrataPrep total RNA microprep kit provides an efficient method to isolate high-quality total RNA from small cell samples. The protocol includes binding of RNA to a solid support, which eliminates the need for organic extraction and alcohol precipitation. The isolated RNA is ready for a variety of quantitative and conventional RT-PCR experiments.

Laser capture microdissection (LCM) enables researchers to collect individual cells from tissue sections.1 This method facilitates the separation of histologically different cells so that proteins, DNA, or RNA can be analyzed from the cells of choice, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. Stratagenes StrataPrep total RNA microprep kit is designed for quick and easy isolation of total RNA from 1 to 5 x 105 cells2 and is particularly useful for isolation of RNA from LCM samples. The resulting RNA can be used for both quantitative and conventional RT-PCR analysis.

RT-PCR Analysis of Varying-Abundance Messages

We isolated total RNA from LCM samples from the core of a human brain tumor section. We used this RNA to synthesize cDNA with the prostar first strand RT-PCR kit. PCR was performed using Stratag enes RT-PCR Primers for Human Gene Transcripts of Varying Abundance Levels according to the instructions.3 RNA of all abundance levels was detected in samples isolated from 200 cells. High abundance RNA was detected in a portion of the RT-PCR product equivalent to 2.5 cells, medium abundance RNA from a portion equivalent to 10 cells, and low abundance RNA from portions equivalent to 13 cells (Figure 1).

In addition, RNA isolated from malignant glioma cells using the StrataPrep microprep kit has been used in differential display experiments. These experiments identified several differentially expressed genes in the invasive rim of the tumor relative to the noninvasive tumor core.4

Conclusions

The StrataPrep total RNA microprep kit is ideal for the isolation of RNA from small cell samples harvested by LCM. The method generates pure, intact RNA with high efficiency that can be effectively amplified by conventional and quantitative RT-PCR.

Acknowledgments

The authors thank Michael Berens, Neurology Research, Barrow Neurological Institute, Phoenix, AZ for providing LCM samples.

REFERENCES

1. Emmert-Buck, M. R., et al. (1996) Science 274: 998-1001.
2. Dolter, K. and Braman J. (2000) Strategies 13: 56-58.
3. Rogers, B. and McKenzie, D. (1998) Strategies 11: 100-102.
4. Berens, M. E., et al. (2000) Proceedings of the American Association for Cancer Research 41:232, Abstract 1477.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Restriction Digests of DNA Isolated with the Perfectprep Gel Cleanup Kit
2. A Better Way to Isolate RNA AND Protein From the Same Sample
3. Isolate It All: siRNA miRNA Total RNA Native Protein
4. Isolate Total RNA and Protein From the Same Sample
5. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
6. Microbial Diversity in Ground and Surface Water Analyzed by Denaturing Gradient Gel Electrophoresis Using the DCode System
7. Analysis of in vitro micronucleus assays using the IN Cell Analyzer 3000 Micronuclei Formation Analysis Module
8. Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 1000
9. The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies
10. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000
11. Identification of cells in S phase using the Cell Proliferation Fluorescence Kit and IN Cell Analyzer 1000
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:5/24/2017)... , May 23, 2017 As Ebola resurfaces ... with four deaths and 20 suspected cases now reported, a ... the PubMed database, showed a correlation between the 2014 and ... Replikin counts rose sharply in 2012-13, which preceded the 2014 ... in the Ebola gene Replikin counts in 2014-15, which again ...
(Date:5/23/2017)... Kong (PRWEB) , ... May 22, 2017 , ... ... third year in a row in the Aragon Research Globe™ for Corporate Learning, ... align with industry direction and market demand, and effectively perform against those strategies. ...
(Date:5/23/2017)... (PRWEB) , ... May 23, 2017 , ... Bacterial biofilms, ... polymeric molecules, can cause diverse pathologies ranging from food poisoning and catheter infections to ... biofilms is in the tens of billions of dollars per year, there is currently ...
(Date:5/22/2017)... ... 2017 , ... Cancer diagnostics and pathology workflow solution provider ... Association for Pathology Informatics Annual Summit at the Wyndham Grand Hotel ... Diagnostic Cockpit and Consultation Portal, Inspirata will present research it led to help ...
Breaking Biology Technology:
(Date:3/29/2017)...  higi, the health IT company that operates the ... , today announced a Series B investment from ... The new investment and acquisition accelerates higi,s strategy to ... population health activities through the collection and workflow integration ... collects and secures data today on behalf of over ...
(Date:3/24/2017)... MILAN , March 24, 2017 The Controller ... Deputy Controller Mr. Abdulla Algeen have received the prestigious international ... Continue Reading ... ... small picture) and Deputy Controller Abdulla Algeen (small picture on the right) ...
(Date:3/23/2017)... , Mar. 23, 2017 Research and Markets ... Market Analysis & Trends - Industry Forecast to 2025" report ... ... at a CAGR of around 8.8% over the next decade to ... report analyzes the market estimates and forecasts for all the given ...
Breaking Biology News(10 mins):