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final exam Flashcards

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1
Q

Why is the RNA isolation protocol much longer than DNA?

A

It is much more unstable and prone to degradation

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2
Q

What is qPCR?

A

Reverse transcription real-time PCR

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3
Q

What occurs in RNAseq?

A

Transcriptome analysis using next-generation sequencing

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4
Q

How is RNA disrupted during isolation?

A

Lytic agent or denaturant (Tri-reagent) must come into contact with the cellular contents when the cells are disrupted

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5
Q

What is the purpose of freezing tissue/cells in liquid nitrogen or on dry ice?

A

Used when tissues or cells are hard, contain capsules or walls, have workflows that prevent immediate interaction of Lytic agent and cellar contents

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6
Q

What occurs in organic solvent extraction?

A

Sample is homogenized in a phenol-containing solution and centrifuged

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7
Q

What is the product of organic solvent extraction?

A

Three layers: lower organic, middle phase that contained denatured proteins and genomic DNA, and upper aqueous phase with RNA

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8
Q

How is RNA collected in organic solvent extraction? Where is it located?

A

Upper aqueous layer, recovered and collected by alcohol precipitation and rehydration

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9
Q

Is nuclease free water used in all RNA applications?

A

Yes!!

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10
Q

What kind of tips are used in RNA applications?

A

Filter

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11
Q

What is the temperature of liquid nitrogen? What is its purpose in RNA isolation? What is the goal of its use?

A

-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

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12
Q

If a gene is in a genome is it always expressed?

A

No, but if it is expressed it gets us closer to phenotype

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13
Q

What environmental factors can alter gene expression (6)?

A

Heat, cold, salt, pathogens, light, nutrient availability

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14
Q

What is transcriptomics?

A

The study of the transcriptome which is the complete set of RNA transcripts that are produced by the genome.

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15
Q

What are 3 differences between RNA and DNA?

A

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

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16
Q

Is everything transcribed in the genome?

A

No

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17
Q

Why are RNA structures unstable?

A

hairpins or self dimers

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18
Q

What are the 3 major classes of RNA? What are their associated percentages of prevalence?

A

mRNA: 1-5%
rRNA: 85%
tRNA: 10%

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19
Q

What are two characteristics of rRNA and tRNA?

A
  1. do not encode for a protein
  2. part of the rRNA is bound to the ribosome during translation (tRNA)
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20
Q

What are two characteristics of mRNA?

A
  1. codes for proteins
  2. has initiation and termination codons
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21
Q

What type of cells is mRNA extensively processed in?

A

Eukaryotic

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22
Q

What are three types of mRNA processing? What are their purposes?

A
  1. 5’ mG7 cap: translation signal, protective
  2. Poly-A tail: protective
    3: Introns splices out: modification while the transcript is being made
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23
Q

What is the difference of pre-mRNA and mature mRNA?

A

Pre-mRNA has exons and introns and goes through capping, splicing, and polyadenylation. mRNA has only cap-exon-poly A tail

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24
Q

What are 3 characteristics of RNA viruses?

A
  1. do not contain DNA
  2. recognized to cause cancers in animals
  3. not suitable for stable integration into host genome
25
Q

How do RNA viruses replicate?

A

By integrating their RNA genome in the host DNA genome- require reverse transcriptase enzyme.

26
Q

What is reverse transcriptase encoded by?

A

RNA genome, polymerase complements mRNA

27
Q

What are retrotransposons? How do they replicate? How were they derived?

A

Eukaryotic genetic elements
Replicate by transcription
Derived from ancient incorporation of viral RNA in our genomes

28
Q

How are retrotransposons integrated back into the genome?

A

Reverse transcription

29
Q

What is the function of RNAi?

A

keeps reverse transcriptase from incorporating and accumulating retrotransposons in genome in varying locations

30
Q

What are oligo dT primers? What are they composed on? Length?

A

Primers that are specific to the poly A tail found on mRNA. Compose entirely of Thymines 18-24bp in length

31
Q

What is the purpose of the oligo dT primer?

A

To bind to and enrich mRNA

32
Q

What is reverse transcriptase?

A

RNA dependent DNA polymerase

33
Q

What is the directionality of reverse transcriptase?

A

Nucleic acid synthesis activity 5’ to 3’

34
Q

What type of strand can reverse transcriptase bind to?

A

Double stranded hybrid

35
Q

What does reverse transcriptase recognize to function?

A

3’ OH group on primer, rest of sequence is negligible. It is not sequence specific

36
Q

What type of activity does reverse transcriptase have?

A

RNAase activity: degrades RNA as it synthesizes a complementary strand of DNA

37
Q

What type of template does reverse transcriptase use? How does it work?

A

Uses single stranded RNA template and recognizes 3’OH of primer and uses dNTPs to create complementary cDNA strand.

38
Q

What are 5 applications and techniques using RNA?

A
  1. RT-PCR (reverse transcriptase PCR)
  2. qPCR (qualitative RT-PCR)
  3. RACE (Rapid Amplification of cDNA Ends)
  4. RNA-Seq (whole transcriptome sequencing)
  5. Microarray (hybridization of whole genome RNA to whole genome cDNA)
39
Q

What are 2 applications of RT-PCR/qPCR?

A

Monitor a single genes expression level in any tissues like developmental time points and cell specific expression. Also monitor expression changes following treatment.

40
Q

What kind of control do you have to include in RT-PCR/qPCR?

A

A housekeeping gene, used to compare/make relative levels of expression

41
Q

What type of template is used in qPCR?

A

cDNA template, more quantitative. Uses fluorescent probe for precise quantification of amount of transcript

42
Q

What are the qPCR results? What do they mean?

A

qPCR give Ct (threshold cycle). Earlier or smaller # cycle means there was more starting amount of cDNA/mRNA

43
Q

What is a threshold cycle (Ct)?

A

The cycle at which the fluorescent signal passes the threshold

44
Q

What is RNA-seq?

A

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

45
Q

What is the number of reads per transcript dependent on (2)?

A
  1. transcript abundance
  2. length of the transcript
46
Q

What type of reagent is used in RNA isolation?

A

Tri-reagent

47
Q

What is the purpose of chloroform in RNA isolation?

A

To separate RNA from DNA

48
Q

What would happen if you had a gDNA contaminant in your RNA isolation sample?

A

Would result in a band as well as the primer would amplify it as well. Lead to false positives

49
Q

What is added to eliminate gDNA in RNA isolation?

A

DNase I

50
Q

What would happen if there was residual DNase I in your RNA isolation sample?

A

Residual enzyme activity would degrade single stranded DNA (primers and synthesized cDNA)

51
Q

What is used in a RNA denaturation and primer annealing cocktail?

A
  1. oligo dT primer
  2. RNA
  3. dNTP mix
  4. RNAase free water
52
Q

What components are used in a cDNA synthesis cocktail?

A
  1. FS buffer
  2. RNAase free water
  3. Reverse Transcriptase (SS III)
  4. 0.1M DTT
53
Q

What components are used in a qPCR cocktail (4)?

A
  1. SybrGreen MM
  2. cDNA (10-20ng)
  3. Forward/Reverse Primer
  4. ddH2O
54
Q

What are the gene/primer pairs for plant anatomy?

A

Flowers- Pistillata (PI)
Leaves- Chaperonin 60- alpha subunit
Stems- Peroxidase 33 (Prx33)

55
Q

What is the housekeeping/control gene/primer pair used in qPCR?

A

Beta- actin

56
Q

What components are in the SYBR green master mix (5)?

A

buffer, dNTPs, thermostable hot start DNA polymerase (AmpliTaq Gold), SYBR green dye, passive reference

57
Q

What is the purpose of passive reference in SYBR green master mix?

A

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.

58
Q

What is the recommended primer concentration in qPCR? How much is added

A

300-800nM. 1uL of each, forward and reverse

59
Q

What is the recommended cDNA concentration in qPCR?

A

10-20ng

molecular genetics (5 decks)

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