Detection of RNA

MOLECULAR & CELLULAR NEUROBIOLOGY

Master Course Cognitive Neuroscience - Radboud University, Nijmegen

INDEX

INTRODUCTION

CELLS AND WITHIN CELLS

IN A NUTSHELL

GENOMICS

MOLECULAR BIOLOGICAL RESEARCH METHODOLOGY

NEURODEVELOPMENT

Chapter 5: Molecular biological research methodology

Molecular biology and Recombinant DNA technology	Detection of DNA, RNA and protein	Generation of gene expression atlases of the CNS
Techniques used in Molecular Biology	Detection of RNA	Gene transfer - transgenic animals
Genetic transmission	In situ hybridization	Optogenetics
Genetic mapping	PCR	Cloning
Genomic and cDNA libraries	Microarray and RNA-seq analysis	Stem cells

Bioinformatics - data analysis

CRISPR-cas genome editing

ChIP-chip/seq

Detection of RNA

Comparison of different methods for quantitation of mRNA

Feature

Northern Blotting

RT-PCR

In Situ Hybridization

Relative and Absolute Quantitation

Yes

Yes

Yes

Detection Limit (Copies of mRNA)

10,000

1 (theoretical)

Medium

Level of Optimization Required

Medium

High

High

Sample Size

30 µg max

Not critical

Tissue sections

Detect Multiple Transcripts

Yes, generally requires stripping and reprobing

Yes, in same tube

No

Localizes mRNA Expression within Tissues/Cells

No

No

Yes

Mapping Studies

No

No

No

Resolve Comigrating mRNAs

No

Yes

No

Sizing mRNAs

Yes

No

No

Detection of Alternatively Spliced Transcripts

Yes

No

No

Distinguish Between Members of Multi-Gene Families

Only if they are different sizes

Only if they are characterized

No

Tolerates Partially Degraded RNA

No

Yes

Yes

Advantages

RNA undergoes minimal processing

Technique can be easily evaluated at various points in procedure

Most sensitive technique available

Localized mRNA within tissues

Tolerates partially degraded RNA

Next page: In situ hybridization Go back to: Detection of DNA, RNA and protein

Feature	Northern Blotting	RT-PCR	In Situ Hybridization
Relative and Absolute Quantitation	Yes	Yes	Yes
Detection Limit (Copies of mRNA)	10,000	1 (theoretical)	Medium
Level of Optimization Required	Medium	High	High
Sample Size	30 µg max	Not critical	Tissue sections
Detect Multiple Transcripts	Yes, generally requires stripping and reprobing	Yes, in same tube	No
Localizes mRNA Expression within Tissues/Cells	No	No	Yes
Mapping Studies	No	No	No
Resolve Comigrating mRNAs	No	Yes	No
Sizing mRNAs	Yes	No	No
Detection of Alternatively Spliced Transcripts	Yes	No	No
Distinguish Between Members of Multi-Gene Families	Only if they are different sizes	Only if they are characterized	No
Tolerates Partially Degraded RNA	No	Yes	Yes
Advantages	RNA undergoes minimal processing Technique can be easily evaluated at various points in procedure	Most sensitive technique available	Localized mRNA within tissues Tolerates partially degraded RNA