final exam Flashcards
Why is the RNA isolation protocol much longer than DNA?
It is much more unstable and prone to degradation
What is qPCR?
Reverse transcription real-time PCR
What occurs in RNAseq?
Transcriptome analysis using next-generation sequencing
How is RNA disrupted during isolation?
Lytic agent or denaturant (Tri-reagent) must come into contact with the cellular contents when the cells are disrupted
What is the purpose of freezing tissue/cells in liquid nitrogen or on dry ice?
Used when tissues or cells are hard, contain capsules or walls, have workflows that prevent immediate interaction of Lytic agent and cellar contents
What occurs in organic solvent extraction?
Sample is homogenized in a phenol-containing solution and centrifuged
What is the product of organic solvent extraction?
Three layers: lower organic, middle phase that contained denatured proteins and genomic DNA, and upper aqueous phase with RNA
How is RNA collected in organic solvent extraction? Where is it located?
Upper aqueous layer, recovered and collected by alcohol precipitation and rehydration
Is nuclease free water used in all RNA applications?
Yes!!
What kind of tips are used in RNA applications?
Filter
What is the temperature of liquid nitrogen? What is its purpose in RNA isolation? What is the goal of its use?
-200ºC, used to freeze the tissue so the cells can be ground and broken while frozen. Goal is to create a fine, dry, and extremely cold powder
If a gene is in a genome is it always expressed?
No, but if it is expressed it gets us closer to phenotype
What environmental factors can alter gene expression (6)?
Heat, cold, salt, pathogens, light, nutrient availability
What is transcriptomics?
The study of the transcriptome which is the complete set of RNA transcripts that are produced by the genome.
What are 3 differences between RNA and DNA?
DNA: double stranded, stable, used to store information (chromosomes), material of inheritance
RNA: single stranded, stable with secondary structures, profiles are dynamic and change with environment
Is everything transcribed in the genome?
No
Why are RNA structures unstable?
hairpins or self dimers
What are the 3 major classes of RNA? What are their associated percentages of prevalence?
mRNA: 1-5%
rRNA: 85%
tRNA: 10%
What are two characteristics of rRNA and tRNA?
- do not encode for a protein
- part of the rRNA is bound to the ribosome during translation (tRNA)
What are two characteristics of mRNA?
- codes for proteins
- has initiation and termination codons
What type of cells is mRNA extensively processed in?
Eukaryotic
What are three types of mRNA processing? What are their purposes?
- 5’ mG7 cap: translation signal, protective
- Poly-A tail: protective
3: Introns splices out: modification while the transcript is being made
What is the difference of pre-mRNA and mature mRNA?
Pre-mRNA has exons and introns and goes through capping, splicing, and polyadenylation. mRNA has only cap-exon-poly A tail
What are 3 characteristics of RNA viruses?
- do not contain DNA
- recognized to cause cancers in animals
- not suitable for stable integration into host genome
How do RNA viruses replicate?
By integrating their RNA genome in the host DNA genome- require reverse transcriptase enzyme.
What is reverse transcriptase encoded by?
RNA genome, polymerase complements mRNA
What are retrotransposons? How do they replicate? How were they derived?
Eukaryotic genetic elements
Replicate by transcription
Derived from ancient incorporation of viral RNA in our genomes
How are retrotransposons integrated back into the genome?
Reverse transcription
What is the function of RNAi?
keeps reverse transcriptase from incorporating and accumulating retrotransposons in genome in varying locations
What are oligo dT primers? What are they composed on? Length?
Primers that are specific to the poly A tail found on mRNA. Compose entirely of Thymines 18-24bp in length
What is the purpose of the oligo dT primer?
To bind to and enrich mRNA
What is reverse transcriptase?
RNA dependent DNA polymerase
What is the directionality of reverse transcriptase?
Nucleic acid synthesis activity 5’ to 3’
What type of strand can reverse transcriptase bind to?
Double stranded hybrid
What does reverse transcriptase recognize to function?
3’ OH group on primer, rest of sequence is negligible. It is not sequence specific
What type of activity does reverse transcriptase have?
RNAase activity: degrades RNA as it synthesizes a complementary strand of DNA
What type of template does reverse transcriptase use? How does it work?
Uses single stranded RNA template and recognizes 3’OH of primer and uses dNTPs to create complementary cDNA strand.
What are 5 applications and techniques using RNA?
- RT-PCR (reverse transcriptase PCR)
- qPCR (qualitative RT-PCR)
- RACE (Rapid Amplification of cDNA Ends)
- RNA-Seq (whole transcriptome sequencing)
- Microarray (hybridization of whole genome RNA to whole genome cDNA)
What are 2 applications of RT-PCR/qPCR?
Monitor a single genes expression level in any tissues like developmental time points and cell specific expression. Also monitor expression changes following treatment.
What kind of control do you have to include in RT-PCR/qPCR?
A housekeeping gene, used to compare/make relative levels of expression
What type of template is used in qPCR?
cDNA template, more quantitative. Uses fluorescent probe for precise quantification of amount of transcript
What are the qPCR results? What do they mean?
qPCR give Ct (threshold cycle). Earlier or smaller # cycle means there was more starting amount of cDNA/mRNA
What is a threshold cycle (Ct)?
The cycle at which the fluorescent signal passes the threshold
What is RNA-seq?
Uses next-generation sequencing (NGS) to study the presence and quantity of RNA in a biological sample at a given moment in time. Compare back to original genome
What is the number of reads per transcript dependent on (2)?
- transcript abundance
- length of the transcript
What type of reagent is used in RNA isolation?
Tri-reagent
What is the purpose of chloroform in RNA isolation?
To separate RNA from DNA
What would happen if you had a gDNA contaminant in your RNA isolation sample?
Would result in a band as well as the primer would amplify it as well. Lead to false positives
What is added to eliminate gDNA in RNA isolation?
DNase I
What would happen if there was residual DNase I in your RNA isolation sample?
Residual enzyme activity would degrade single stranded DNA (primers and synthesized cDNA)
What is used in a RNA denaturation and primer annealing cocktail?
- oligo dT primer
- RNA
- dNTP mix
- RNAase free water
What components are used in a cDNA synthesis cocktail?
- FS buffer
- RNAase free water
- Reverse Transcriptase (SS III)
- 0.1M DTT
What components are used in a qPCR cocktail (4)?
- SybrGreen MM
- cDNA (10-20ng)
- Forward/Reverse Primer
- ddH2O
What are the gene/primer pairs for plant anatomy?
Flowers- Pistillata (PI)
Leaves- Chaperonin 60- alpha subunit
Stems- Peroxidase 33 (Prx33)
What is the housekeeping/control gene/primer pair used in qPCR?
Beta- actin
What components are in the SYBR green master mix (5)?
buffer, dNTPs, thermostable hot start DNA polymerase (AmpliTaq Gold), SYBR green dye, passive reference
What is the purpose of passive reference in SYBR green master mix?
Includes a proprietary version of ROXTM dye, an internal passive reference, to normalize non- PCR–related fluorescence fluctuations and to minimize well-to-well variability that result from a variety of causes, such as pipetting error and sample evaporation.
What is the recommended primer concentration in qPCR? How much is added
300-800nM. 1uL of each, forward and reverse
What is the recommended cDNA concentration in qPCR?
10-20ng
