RNA EXTRACTION (CANDIDA ALBICANS/NPS/OPS)

Hot acidic phenol

RNA extraction using Candida albicans preparation of yeast RNA by extraction with ______

Intact cells

Yeast RNA can be isolated efficiently and directly from ____

1. Acidic phenol (pH 5)
2. SDS at 65C

Yeast RNA can be isolated efficiently and directly from intact cells by extraction with (2):

Grow yeast cells in 10mL of desired medium to mid-exponential phase (OD 600 = 0.5)

1. Grow yeast cells in ____ of desired medium to mid-exponential phase

Transfer culture to 50-ml centrifuge tube and centrifuge cells 3 min at 15,000g, 4°C.

2. Transfer culture to 50-ml centrifuge tube and centrifuge cells ___ min at 15,000g, 4°C.

Discard supernatant, resuspend pellet in 1 ml ice- cold water.
Transfer to a clean 1.5-ml microcentrifuge tube.
Microcentrifuge 10 sec at 4°C, and remove supernatant.

3. Discard supernatant, resuspend pellet in _________.
Transfer to a clean 1.5-ml microcentrifuge tube.
Microcentrifuge 10 sec at 4°C, and remove supernatant.

Resuspend cell pellet in 400 μl TES solution.
Add 400 μl acid phenol preheated to 65oC and vortex vigorously 10 sec.
Incubate 15 min at 65°C with brief vortexing for 10 sec every 5 min interval

4. Resuspend cell pellet in _____.
Add 400 μl acid phenol preheated to 65oC and vortex vigorously 10 sec.
Incubate 15 min at 65°C with brief vortexing for 10 sec every 5 min interval

Place on ice for 2 min. Microcentrifuge 5 min at top speed, 4°C.

5. Place on ___ for ___. Microcentrifuge 5 min at top speed, 4°C.

Transfer aqueous phase to a clean 1.5-ml microcentrifuge tube and add 400 μl chloroform.
Vortex vigorously and microcentrifuge 5 min at top speed, 4°C.

6. Transfer aqueous phase to a clean 1.5-ml microcentrifuge tube and add ________
Vortex vigorously and microcentrifuge 5 min at top speed, 4°C.

Transfer aqueous phase to a new tube, add 40 μl of 3 M sodium acetate, pH 5.3, and 1 ml of ice-cold 100% ethanol and precipitate. Microcentrifuge 5 min at top speed, 4°C.

7. Transfer aqueous phase to a new tube, add ______, pH 5.3, and 1 ml of ice-cold 100% ethanol and precipitate. Microcentrifuge 5 min at top speed, 4°C.

Wash RNA pellet by vortexing briefly in ice-cold 70% ethanol. Microcentrifuge as before to pellet RNA.

8. Wash RNA pellet by vortexing briefly in _____. Microcentrifuge as before to pellet RNA.

Resuspend pellet in 50 μl H2O.

9. Resuspend pellet in ______

Absorbance 260/280

10. Read Absorbance at ___

Candida albicans

Organism used in the RNA extraction procedure

Acidic phenol

Reagent used to isolate RNA from yeast cells

65C

Temperature at which phenol is preheated for the extraction process

TES

Buffer solution used to resuspend the cell pellet

5

The pH of acidic phenol used in the extraction

15,000xg

Centrifugation speed in g-force used to initially pellet yeast cells

Chloroform

Chemical added after phenol extraction to separate aqueous and organic phases

Sodium acetate

Chemical solution used for RNA precipitation

70%

Concentration of ethanol used to wash the RNA pellet

50ul

Final volume of water used to resuspend the RNA pellet

Mid-exponential

Optimum growth phase for yeast cells used in extraction

RNA stabilization and solubilization

Why 1ml ice cold water is used to resuspend the pellet?

Buffer for cell suspension

Function of TES solution

Cell lysis and protein denaturation

Function of 400ul acid phenol

Phase separation

Function of 400ul chloroform

RNA precipitation

Function of ice-cold 100% ethanol

RNA washing

Function of ice-cold 70% ethanol

500uL of QIAzol

1. Aspirate _____ reagent on your microcentrifuge tube Note: 500 ul of Phenol reagent is Sufficient for 100,000-800,000 volume It the sample.

NPS/OPS

2. Add an equal volume of ______ to QIAzol.

100 ul

3. Add _____ of chloroform

4. Vortex mix for 15 seconds.

5. Incubate @ RT for 3 mins

6. Centrifuge for 12, 000 rpm for 10 mins

7. Transfer upper aqueous phase into new Microcentrifuge tube (200 uL)

100 ul

In 2nd homogenization, add _____ of Chloroform

1. Isopropanol
2. NA Acetate

These 2 reagent are used for RNA precipitation:

10. Transfer RNA containing upper aqueous phase approximately 200ul on a new MCT.

11. Add 250 ul of isopropanol and NA Acetate

12. Vortex mix 10-20 times

13. Let sit for 5 min @ RT.

14. Centrifuge for 12,000 RPM for 5 mins à Pellet formation (discard Supernatant)

15. Add 100ul of 75% ETOH to pellet.

16. Centrifuge for 7500 rpm for 3 mins.

17. Remove Supernatant / Decant

18. Repeat step 15 - 17.

RNA Precipitation Steps using NPS/OPS

19. Heat tubes open @ 65C for 5-10mins

20. Add 100ul of Grade Water -> Vortex Mix

21. Store @ -70 to -80C till further use

RNA elution Steps using NPS/OPS

QIAzol

Reaagent used for inital homogenization in NPS/OPS

100ul

Volume of chloroform added during both homogenization steps

Aqueous phase

Phase transferred to a new microcentrifuge tube after centrifugatio

Isopropanol

Chemical added to the RNA containing phase for precipitation

Sodium acetate

Additional chemical added with isopropanol to aid RNA extraction

75%

Concentration of ethanol used for washing RNA in NPS/OPS

Washing

Function of 75% Ethanol in NPS/OPS

-70 to -80C

Storage temperature for the final RNA preparation

100ul

Final volume of elution in RNA in NPS/OPS

Precipitation

Function of isopropanol in RNA (NPS/OPS)

Precipitation

Function of NA acetate in RNA (NPS/OPS)

Washing

Function of 75% Ethanol in RNA (NPS/OPS)

Homogenization

Function of Chloroform in RNA (NPS/OPS)

100,000-800,00 volume

500ul Phenol reagent is sufficient for ______

100% ethanol precipitates RNA, while 70% ethanol washes away impurities without dissolving the RNA. Omitting 70% ethanol could result in residual salts and contaminants that affect RNA purity.

What is the purpose of using both 100% and 70% ethanol in RNA extraction, and what would happen if 70% ethanol was omitted?

RNA is more susceptible to degradation by RNases, which are ubiquitous and stable enzymes. Precautions include using RNase-free reagents, wearing gloves, and keeping samples on ice to prevent enzymatic activity.

Why is RNA often less stable than DNA during extraction, and what additional precautions are needed to ensure RNA integrity?

RNA precipitation

What is the primary function of 100% ethanol in the RNA extraction procedure?

To wash away impurities

Why is 70% ethanol used after 100% ethanol in RNA extraction?

Chloroform

Which reagent is added to separate the aqueous and organic phases during RNA extraction?

Cell lysis and protein denaturation

What is the role of acidic phenol in the RNA extraction procedure?

Buffer the cells

TES solution in RNA extraction serves to:

Phenol or guanidine

In RNA extraction, which contaminant is most likely indicated by a low A260/A230 ratio?

To prevent RNA degradation

What is the primary purpose of keeping cells on ice during RNA extraction?

2.0-2.2

A pure RNA sample typically has an A260/A280 ratio of:

Neutralize cell membrane charges

During the preparation of competent cells, CaCl₂ is used to:

High quality reagents

If RNA extracted from Candida albicans shows no detectable signal, which is NOT a likely cause?

Sodium acetate and isopropanol

What reagent combination is used for RNA precipitation in most RNA extraction protocols?

To make cells more permeable to plasmid DNA

In bacterial transformation, what is the purpose of the heat shock step?

Contamination with proteins

If the A260/A280 ratio of RNA is significantly lower than 2.0, what might this indicate?

Young cultures produce less RNA

If RNA yields are low when using young cultures instead of mid-exponential cultures, the most likely reason is:

Using RNase-free reagents

Which of the following would improve RNA quality if degradation is observed in extracted samples?