Download 16 RNA extraction

Today
• House Keeping
–CARC?
• RNA extraction, DNAse treatment
• Quality Control: Bioanalyzer
• Stay on schedule, breaks are for:
• Background
• ppt
Trizol reagent
• Isolates high quality total RNA (as well as
DNA and proteins)
Sequential precipitation of RNA, DNA, and
proteins from a single sample
Maintains the integrity of the RNA due to highly
effective inhibition of RNase activity
• Careful!
Phenol (toxic and corrosive)
Guanidine isothiocyanate (irritant)
ITINERARY
• HANDOUT
1-7 spin at same time
• 10 minutes : background
– 8-11 spin at same time
• 15 minutes : background
– 12-14+15 incubate then spin at same time
• 20 minutes :
– 16-20-21 air dry re-suspend
• 5 minutes :
– 21 resuspend, T1-2 incubate
• 30 minutes : 5 min break; ppt.
– T3-6
• DEMO bioanalyzer 45 min
• Start RT rxns
Central Dogma of Molecular Biology
Central Dogma of Molecular Biology
Central Dogma of Molecular Biology
DNA
RNA
Reverse Transcriptase (RT)
Using Physella acuta to Determine the Predictive
Value of Biomphalaria glabrata as a Model for
Gastropod Immunity
Planorbidae
Gastropod
Immunity
B. glabrata
Physidae
P. acuta
Immune
factors
-LBP/BPI
-MIF
-Biomphalysin
-Aplysianin
-PGRP
-FREPs
?
Septic
Exposure
Workflow
454 RNA-Seq
reads
Contig
remove short contigs
(<500nt) & merge
assemblies in
Sequencher
REFERENCE
TRANSCRIPTOME
12,645 contigs
Baseline
Targeted Analysis of Immune Factors
•
•
The reference transcriptome assembled in silico from P. acuta RNAseq data
was screened for the presence of particular immune factors, previously
characterized from B. glabrata.
The biological reality of in silico predicted transcripts of immune factors will be
validated experimentally via (sequencing of) RT-PCR amplicons.
B. glabrata
Expression in
Immune factors
P. acuta
Biomphalysin
Aplysianin/Achacin
PGRP
LBP/BPI
FREP
Yes
?
?
?
Yes
A typical bacterium contains 0.05–0.10 pg of RNA, making up about 6% of its total weight. A mammalian cell, being much larger, contains more
RNA, 20–30 pg in all, but this represents only 1% of the cell as a whole (Alberts et al., 1994). It is important to appreciate that not all of this RNA
constitutes the transcriptome. The latter is just the coding RNA - those molecules that have been transcribed from protein-coding genes and which
are therefore capable of being translated into protein. Most of the cellular RNA does not fall into this category because it is non-coding.
Non coding RNA is more diverse than the coding RNA and comprises transcripts with a number of different functions, all of which are performed by the RNA molecules
themselves. In both prokaryotes and eukaryotes the two main types of non-coding RNA are:
Ribosomal RNAs (rRNAs), which are the most abundant RNAs in the cell, making up over 80% of the total in actively dividing bacteria. These molecules are components
of ribosomes, the structures on which protein synthesis takes place.
Transfer RNAs (tRNAs) are small molecules that are also involved in protein synthesis, carrying amino acids to the ribosome
Ribosomal and tRNAs are present in the cells of all species. The other non-coding RNA types are more limited in their distribution (see Figure 3.3 ). Eukaryotes, for
example, have a variety of short non-coding RNAs that are usually divided into three categories, the names indicating their primary locations in the cell:
Small nuclear RNA (snRNA; also called U-RNA because these molecules are rich in uridine nucleotides), which is involved in mRNA processing (Section 10.1.3);
Small nucleolar RNA (snoRNA), which plays a central role in the processing of rRNA molecules (Section 10.3.1);
Small cytoplasmic RNA (scRNA), a diverse group including molecules with a range of functions, some understood and others still mysterious.
NON-CODING RNA (ncRNA; e.g Yasuda J, Hayashizaki Y. The RNA continent. Adv Cancer Res. 2008;99:77-112.
Volume 157, Issue 1, p77–94, 27 March 2014
Why is this good RNA?
$15,000.
WHAT IS IT?
•Cutting edge Lab-on-a-Chip Products
Agilent Technologies developed microfluidic Lab-on-a-Chip technology. This
technology utilizes a network of channels and wells that are etched onto glass or
polymer chips to build mini-labs. Pressure or electrokinetic forces move pico liter
volumes in finely controlled manner through the channels. Lab-on-a-Chip enables
sample handling, mixing, dilution, electrophoresis and chromatographic
separation, staining and detection on single integrated systems. The main
advantages of Lab-on-a-Chip are ease-of-use, speed of analysis, low sample and
reagent consumption and high reproducibility due to standardization and
automation.
•What is the detection principle of the bioanalyzer system?
The bioanalyzer instrument detects biomolecules by laser-induced fluorescence.
During chip preparation, a dye concentrate is mixed with a gel. With the help of
the priming station (a syringe), the channels of the chip are filled with the gel-dye
mix. During the chip run, the dye intercalates directly with the analytes (DNA and
RNA assays) or with SDS-micelles (protein assays). When analyzing cells they need
to be pre-stained with application specific dyes.
TYPICAL OUTPUT FORMAT
Ratio = RN number
For quality!
http://www.nature.com/jid/journal/v127/n2/full/5700557a.html
PARASITES AND SNAIL BIOLOGY
DNA
“identity, possibilities”
phylogenetics
RNA
“intentions”
transcriptomics
CTAB
Trizol
gel electrophoresis
nanodrop spec
Bioanalyzer
DNA-free,
PCR
rDNA/mito
TA cloning, B/W screening
electrophoresis
direct sequencing
Sequence ID (BLAST)
editing
+
Sevilleta snails
Phylogenetics
GenBank
submission
Qiagen plasmid extraction
Restriction digests
M13 sequencing
Primer design, walking
RT-PCR
gel
Protocol on-line
http://biology.unm.edu/cmadema/ThermoScriptRTPCR.pdf
https://lifescience.roche.com/shop/PrintView?langId=-1&storeId=15016&articleId=68045
MM-denat
MM-RT
TYPICAL OUTPUT FORMAT
Ratio = RN number
For quality!
http://www.nature.com/jid/journal/v127/n2/full/5700557a.html