Experimental Techniques

Question 1

Lysis into nuclear and cytoplasmic cellular fractions?

Answer 1

1. Cells washed in PBS
2. Cell Lysis Buffer: 0.5% Nonidet P-40 (milder, non-ionic detergent), 10mM HEPES (zwitterionic organic chemical buffer reagent –> maintains physiological pH –> maintains enzyme structure and function), 10mM KCl, 0.1 mm EDTA (salt), 1mm DTT (dithiothreitol), 0.5mM pMSF (serine protease inhibitor), protease inhibitor cocktail
   - -> on ice, 15-20min for cells to swell, vortex, centrifuge
   * Nonionic detergent –> mimic internal conditions of the cell to preserve the structure/function of the contents

OR 10mM Tris-HCl (salt –> acidity/osmolarity); 150mM NaCl, 1.5 mM MgCl2, 0.65% Nonidet-40 detergent (Triton X-100, DO NOT lyse the membrane), RNase inhib.

Detergents: RIPA - ionic (harsh); NP-40/Triton X-100; CHAPS (zwitterionic/better at solubization)

3. Pelleted nuclei washed with cell lysis buffer –> nuclear extraction buffer: 20mM HEPES, pH 7.5; 400mM NaCL, 1mM EDTA, 1mM DTT, 1mM PMSF; Protease inhibitor cocktail (30’ on ice)
4. Nuclear extraction –> -70C

Question 2

Trizol reagents

Answer 2

Acid guanidinium thiocyanate-phenol-chloroform extraction

• Phase separation by centrifugation of a mixture of the aqueous sample and a solution containing water saturation chloroform and phenol –> upper aqueous phase and a lower organic phase
• Guanidin thyocyanate –> protein denaturation (chaotropic agent = disrupts hydrogen bonds): denatures proteins, RNAse, separates RNA from ribosomal proteins
• Nucleic acids –> aqueous phase
• Proteins –> organic phase

The pH determines which nucleic acids get purified: acidic (pH 4-6)–> DNA into the organic phase, RNA remains sin the aqueous phase

Phenol: DISADVANTAGE = despite pure RNA –> desalted and concentrated with an alcohol precipitation step –> does not quantitatively recover small nucleic acid molecules

OR SOLID-PHASE EXTRACTION: large RNAs in the presence of high concentration of denaturing salts –> do not effectively recover small RNAs

Question 3

miRVana

Answer 3

1. Sample lysed in a denaturing lysis solution which stabilizes RNA and inactivates RNAses: LYSIS and DISRUPTION –> lysis solution and homogenate additive
2. Organic extraction –> acid-phenol chloroform –> semi-pure RNA sample
3. Purification over glass fiber filter –> solution formulated for miRNA retention
   - TOTAL RNA: ethanol 1.25vol, washes, elution
   - SMALL RNA (<200nt) - 100% EtOH (25% ethanol samples); small RNA eluted; 55% ethanol samples - small RNA sticks to the glass

Question 4

Reverse trasncription mature miRNA

Answer 4

Taqman microRNA reverse transcription kit

1. Stem loop RT (steric hindrance so cannot anneal to pro- or pre-miRNA) –> Thermocycler: 30’ 16C, 30’ 42C, 5’ 85C, 4C
2. Real Time PCR:
   - Forward primer (tail. i.e. extra 5’ nt= increase Tm depending on miRNA sequence composition)
   - Reverse primer (universal primer complementary to the sequence within the RT stem-loop primer)
   - Dye-labeled taqman probe (Q/F); MGB = minor groove binder, formation of stable duplexes with ss DNA targets, increases Tm for shorter probes

2^(-dCt); dCt = Ct(miRNA)-Ct(U6)

Question 5

Reverse Transcription platforms?

Answer 5

1. Universal tailing primers or random hexamers: polyadenylase to generate poly-A tails and an anchored poly-T primer –> cDNA –> (False positive?)
2. Unique, sequence specific RT primers for cDNA –> increased specificity

Question 6

Real-Time PCR methods?

Answer 6

Taqman: Fluorogenic-labeled probes that use 5’ nuclease activity of Taq DNA Polymerase –> 5’ to 3’ nucleolytic activity of the DNA pol. cleaves the hybridized fluorogenic probe btw. the reporter dye and the quencher dye –> fragments of reporter dye displaced –> increased fluorescence (FRET: Fluorescence Resonance Energy Transfer)

SYBR Green: binds the minor groove of dsDNA –> intensity of fluorescence increase –> does not distinguish btw primer dimers so may cause overestimation

Question 7

LNA-Flow FISH

Answer 7

LNA: nucleic acid analog - ribose sugar constrained by a methylene bridge btw 2’oxygen and 4’carbon –> N-type conformation ; LNA bases linked by the native phosphate backbone found in DNA and RNA –> allows synthesis of DNA and RNA that contain LNA monomers; need shorter hybridization times; better signal to noise ratio than DNA counterparts (DNA oligont probes); increase in Tm 1-8C (DNA), 2-10C RNA; 20-25 nt probes, Tm ~85-90C

LNA fluorescent in situ hybridization followed by rolling-circle amplification

• 10 fluidically isolatable chambers - Cell Tak solution –> PBS
  1. Cells fixed onto the chamber and permeabilized (2000 cells/chamber)
  2. Diethyl Pyrocarbonate Treatment –> inactivates RNAses by carbethoxylation and decreases background signal (in bacteria)
  3. Permeabilization?
  4. miRNA-155 LNA probe with dioxygenin happen immunotag - hybridization
  5. Cells blocked with 2% BSA
  6. Duolink mouse PLUS and MINUS probes anti-DIG ab
  7. Ligated with 2 additional oligonucleotides to forma circular template for RCA with Phi29 bacterial polymerase
  8. FITC labeled oligonucleotide probes complementary to the ligated circular template are hybridized with the resultant circular concatamers

Controls: (i) no LNA - LNA probe not added during hybridization; (ii) ‘no dye’ control - LNA probe is hybridized to the target sRNA but hybridization not detected due to the absence of fluorescent stain; (iii) ‘non-expressed sRNA control’ - LNA probe that targets a non-existent or non-expressed sRNA (monitor non-specific hybridization)

Question 8

RNA-Chip analysis

Answer 8

• Buffers contain RNAse inhibitors
• Isolate nuclei –> IP with a-p53, anti-p68, anti-Drosha–> wash–> elution–> RNA:DNA pellets–> nuclease free water+1M Tris-HCl pH=7.5 + 50mM MgCl2+DnaseI (30’, 37C) –> extracted, phenol/chloroform (5:1) and ppt with ethanol –> dissolve in water

Question 9

Nuclear Run-On Assay

Answer 9

• An aliquot of PCR fragment dot blotted onto pos. charged nylon –> RNA probes

To test in vitro pri-miRNA processing –> radiolabeled pri-miRNA with immunoprecipitated Flag-tagged Drosha complex

• Cells infected with retroviral constructs carrying fusion genes encoding enhanced GFP and a short segment of pro-miRNA (decrease in GFP, high processing)

Question 10

i.d. vs. s.c. B16 melanoma injection

Answer 10

Influences tumorogenicity and immunogenicity

s. c. –> less DCs and injection does not induce inflammation
i. d. –> more DCs, greater CD8+ T cell infiltration

Question 11

What is LNA-Flow FISH?

Question 12

What is Rolling Circle Amplification?

Answer 12

• To amplify the LNA probe bound miRNA signal –> improved signal specificity
  1. DNA polymerase
  2. A short DNA primer
  3. A circulating DNA template
  4. Deoxynucleotide triphosphates
• -> PLUS and MINUS duolink probes with short strand of DNA –> Two circle forming DNA oligonucleotides (connector oligos)–> Ligation –> Polymerase amplification –> Hybridization of labeled complementary oligonucleotieds –> Fluorescent microscopy

Question 13

What is Digoxygenin

Answer 13

• Purchase LNA double DIG-labeled probes miR-155 and scrambled
• Hapten; all purpose immuno-tag –> specificity

Question 14

Other miRNAs expressed in T cells?

Answer 14

miRNA-146a unregulated during cytokine exposure;

miR-130 and miR-301 upregulated

Question 15

Most common RT-PCR pitfalls?

Answer 15

1. Poor primer and probe design: primer/probe Tm, complementarity, and secondary structure
2. Poor quality RNA: Degraded or impure RNA
3. Contamination - no template control should be included
4. No RT control: accounts for carry over genomic DNA
5. Normalization control
6. Dissociation (Melting) curves not provided by SYBR green; expected: sharp peak at the Tm of the amplicon, , NAC and NTC will not generate significant fluorescent signal –> SPECIFIC PRODUCTS
7. Not setting the baseline and threshold properly:
8. Poor efficiency of the reaction: Eff = 10 (–1/slope) – 1; 90–110% ( 3.6 > slope > 3.1); Factors that influence efficiency: length of the amplicon, secondary structure, and primer design
9. Inappropriate range of standard curves

Question 16

What are the microRNA precursor and mature transcript controls?

Answer 16

Proposed: 28S for pri-miRNA, 5S for pre-miRNA, 18S for m-miRNA

5S (120 nt) - pre-miRNA
18S (>1kb) - pri-miRNA
U6 snRNA (150nt) - m-mRNA

Normalization paper:

Question 17

How to calculate power?

Answer 17

1. Effect size of interest
2. Standard Deviation
3. Chosen significance level (1.96)
4. Chosen power (0.84)
5. Alternative hypothesis
6. Sample size

Question 18

What is power?

Answer 18

The probability that the null hypothesis will be rejected when it is false

Question 19

What is null hypothesis?

Answer 19

The default ‘position’ that no difference exists between experimental and control groups.

Question 20

What is alternative hypothesis?

Answer 20

Defines the relationship that a researcher believes to be true (i.e. greater tumor growth in experimental group than controls)

Question 21

What is effect size?

Answer 21

When only two groups compared the effect size is the difference in means, D=(difference in means)/(SD)
- Expected mean difference between experimental and control groups

Question 22

What is the significance level?

Answer 22

The chance of obtaining a false positive result due to sampling error (known as a Type I error). It is usually set at 5%

Question 23

What is siRNA?

Answer 23

Small interfering RNA (siRNA). dsRNA with 2 nt 3’ end overhangs that activate RNAi, leading to the degradation of mRNAs in a sequence-specific manner dependent upon comp’limentary binding of the target mRNA.

Question 24

What is shRNA?

Answer 24

Short hairpin RNA (shRNA) that contains a loop structure that is processed to siRNA and also leads to the degradation of mRNAs in a sequence-specific manner dependent upon complimentary binding of the target mRNA.

Question 25

HIF1a specific shRNA lentivirus

Answer 25

• ## puromycin resistance gene for the selection of cells stably expressing shRNA –> transfection

Question 26

Simplest RNAi method?

Answer 26

Cytosolic delivery of siRNA oligonucleotides –> limited to cells capable of transfection and is primarily utilized during transient in vitro studies; variable transfection efficiencies

RNAi = sequence-specific degradation of host mRNA through the cytoplasmic delivery of double-stranded RNA (dsRNA) identical to the target sequence

The proper selection of a target sequence for a given gene of interest remains one of the most critical components of successful gene knockdown

Question 27

shRNA mediated RNAi?

Answer 27

Another form of RNAi involves the use of short hairpin RNAs (shRNAs) synthesized within the cell by DNA vector-mediated production

shRNAs are capable of DNA integration and consist of two complementary 19–22 bp RNA sequences linked by a short loop of 4–11 nt similar to the hairpin found in naturally occurring miRNA

Used to generate stable knockdown cell lines, thereby eliminating the need for multiple rounds of transfection and greatly increasing reproducibility of results.

Question 28

Choice between siRNA and shRNA?

Answer 28

Depends on several factors such as cell type, time demands, and the need for transient versus stable integration.

Question 29

Disadvantages of siRNA use?

Answer 29

• Off-target effects due to the high concentration of cytoplasmic siRNA
• As the cells divide, the siRNA concentration becomes diluted, thereby rendering the generation of a long-term cell line with the desired target gene knockdown unfeasible

Question 30

Constructing shRNA vectors?

Answer 30

oligonucleotide-based cloning and PCR-based cloning

Question 31

Method of vector transduction?

Answer 31

Lentiviral-mediated transduction provides a convenient method of introducing shRNA into dividing or non-dividing cells and, in general, is less toxic to the cells than adenoviral-mediated transduction

Functional assay to confirm knockdown

Question 32

Selecting shRNA target site?

Answer 32

Determine whether the gene of interest has one or multiple splice variants. Decide whether you want to target all potential forms of a gene or specific splice variants.

Question 33

What are lentiviral vectors?

Answer 33

Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell.

Question 34

What is the sample size for the experiments proposed?

Answer 34

In order to achieve a 50% effect size (expected difference between means, 10X decrease in control group vs. 15X decrease in the experimental group) with 20% SD (3X SD) –> need 6 mice/group

Question 35

ANOVA?

Answer 35

Analysis of variance (prevent the chance of type I error)
- provides a statistical test of whether or not the means of several groups are equal

Follow up tests: in order to assess which groups are different from which other groups or to test various other focused hypotheses

Tukey’s range test: to compare every group mean with every other group mean and typically incorporate some method of controlling for Type I errors; standardized range distribution

Question 36

Type I error

Answer 36

False positive: incorrect rejection of a true null hypothesis

Question 37

Type II error

Answer 37

False negative: failure to reject a false null hypothesis

Question 38

Biological control for HIF1a mediated expression in T cells?

Answer 38

GLUT1, Phosphofructokinase

Question 39

Choosing siRNA sequence?

Answer 39

1, Avoid GC rich sequences (> 50%).
2, Avoid stretches of 4 or more nucleotide repeats.
3, Avoid sequences homology with other related or unrelated genes

Question 40

Adenoviral trunsduction?

Answer 40

Appropriate amount critical for experimental success
- A range of 10-200 MOI (multiplicity of infection) is used for most cell lines
- Infectivity particles are measured in culture by a plaque-forming unit assay (PFU) that scores the number of viral plaques as a function of dilution.
-

Question 41

An inverse reporter for detecting miR-132 expression in vivo (B. Luikart)

Answer 41

• Inverse miRNA-132 reporter lentivirus by placing two perfectly complementary miR-132 target sequences downstream of mCherry driven by an internal ubiquitin promoter
• Reporter control: placed the reverse complement of the miR-132 target downstream of mCherry
• retroviral miR-132 sponge with four perfect miR-132 targets downstream of the U6 promoter and with EGFP downstream of the ubiquitin promoter

Question 42

B. Luikart paper qRT-PCR

Answer 42

pri-miRs –> random hexamer primers
One sample serially diluted –> standard curve –> SYBR green

m-miRNA –> Taqman microRNA Assay ; standard curve

Question 43

siRNA design

Answer 43

siRNA sequences between 19–29 nt are generally the most effective. Sequences longer than 30 nt can result in nonspecific silencing. Ideal sites to target include AA dinucleotides and the 19 nt 3’ of them in the target mRNA sequence. Typically, siRNAs with 3’ dUdU or dTdT dinucleotide overhangs are more effective . More recent data indicates that other dinucleotide overhangs maintain activity, however siRNAs can be cleaved by RNase at single-stranded G residues and therefore GG overhangs should be avoided. Choose at least 2–4 target sequences at different positions since mRNAs tend to be highly structured and bound by regulatory proteins. Avoid designing siRNAs with a 4–6 poly(T) tract, since it acts as a termination signal for RNA pol III.
Check to make sure your siRNA sequences do not have homology to other coding sequences.
Sequences should have a G/C content between 35–55%.

Question 44

shRNA design

Answer 44

should consist of the sense and antisense sequences (each 19–21 nt in length) separated by loop structure, and a 5’ AAAA overhang. When designing the loop structure, Ambion scientists and others recommend using a 9 nt spacer (TTCAAGAGA), while Invivogen uses a 7 nt loop (TCAAGAG) for certain vectors, though this can vary depending on your system. Loop sequences of 3 to 9 nt in length have been shown to be effective. Additionally, when creating the shRNA cassette, they suggest that the sense strand come first, followed by the spacer and then the antisense strand. 5’ overhangs in the shRNA construct should be avoided, as they could result in silencing of the shRNA. In addition to manually designing an siRNA or shRNA, there are also several design programs available. A few of them are included in Table 2. Additionally, there are multiple companies that offer premade siRNAs and shRNAs

Question 45

siRNA/shRNA controls

Answer 45

To ensure that the effects observed after RNAi treatment are the result of gene silencing and not merely the introduction of the siRNA/shRNA or from activation of RNAi pathways, it is important to include the appropriate controls (Table 3). The two most common controls are scrambled and non-targeting controls. A scrambled control is exactly what it sounds like, it involves taking the siRNA or shRNA sequence and randomly rearranging its nucleotide sequence. A non-targeting control, on the other hand, is an siRNA/shRNA sequence designed such that it does not target any known genes in the target organism. These controls activate the RNAi machinery and allow baseline determination of the effect of introduction of duplex RNA on gene expression. However, it should be noted that even non-targeting siRNA controls induce a stress response within cells . While both of these types of control sequences will be incorporated into Dicer and activate the RNAi pathway, it is possible that the scrambled control may target an unintended mRNA. As such, care should be taken in designing the scramble sequence to make sure it follows the guidelines mentioned above, and does not target another mRNA sequence.

For shRNAs other important controls include an empty vector control, which contains no shRNA insert, allowing for the determination of the effects of transfection/transduction on gene expression and the response of the cell. Finally, untreated cells (no transfection or transduction) provide a reference to compare the other cells to. This will allow you to determine the cytotoxicity of your particular siRNA delivery method.