2. RNA Isolation and qRT-PCR

We have developed and extensively tested this protocol for qRT-PCR from whole adult flies. For each experimental condition (infected/uninfected/sterile injury, different genotypes, etc.) we use at least three biological replicates (more is often better).

Where specific vendors and product numbers are included, we have tested others and found them to work worse in this protocol.

Nucleic Acid Extraction:

Requirements: Trizol, Chloroform, Isopropanol, 75% EtOH

1. Homogenise 3 flies/sample in 100μl of Tri Reagent (TriReagent from Sigma, Trizol from Life Technologies, or whatever). Note that although we use 3 flies per sample whenever possible, this protocol works reliably with as little as 1 fly per sample.

2. Add 20 μl chloroform. Mix thoroughly. Incubate 3 mins at room temperature.

3. Centrifuge samples at 4°C, 12000g or higher, for 15 mins.

4. Decant the aqueous phase (approximately 60 μl) into a new tube.

5. Add 50 μl isopropanol to the Eppendorfs containing aqueous phase of samples. Mix thoroughly and incubate at room temperature for 10 minutes.

6. Centrifuge the samples at 4°C, 12000g or higher, for 10 minutes.

7. Discard the supernatant and add 200 μl 75% EtOH.

NOTE: If you want to store the RNA, it is very stable under ethanol: you can keep these pellets in the -20° for months. If you resuspend it, be sure you are ready to go all the way to reverse transcription. 

DNAse treatment:

Requirements: ultrapure water, DNAse buffer, DNAse I, EDTA  

1. Spin samples at room temperature, max speed, 5 minutes. 

2. Once the 5 minute spin is done, remove EtOH with a pipet. There should be a visible pellet—don’t discard it.

3. Quick-spin and remove EtOH again. Your RNA pellet should still be damp but there should be almost no liquid remaining in the tube.

4.  While this spin is going on, make up DNAse mix: per sample: 

   34 μl ultrapure water 

   4 μl 10x DNAse buffer (Thermo Fisher 10202730)

   2 μl RNAse-free DNAse I, 1 U/μl (Thermo Fisher 10649890)

   (be sure to make a little bit extra—usually add 10% to all the volumes) 

5. Add 40 μl DNAse mix to each tube. The pellets should dissolve easily and rapidly; no mixing should be necessary.

6. Incubate at 37° for 30 min. 

7. Add 4 μl 25 mM EDTA. 

8. Incubate at 65° for 10 min.

9. Freeze at -80° to store if necessary, but if you have the time it’s better to go straight to reverse transcription. 

Reverse transcription: 

Requirements: random hexamers, RT buffer, RiboLock, reverse transcriptase, dNTPs 

1. To 10 μl DNAse-treated RNA add 1 μl random hexamers (0.2 mg/ml) (Thermo Fisher 10580091)

2. Incubate at 70° for 5 min

3. Put directly on ice. Once they’re cold, quick-spin the tubes to get everything at the bottom, then put them back on ice.

4. Add to each sample a mix containing:

   4 μl 5x RT buffer (comes with enzyme)

   1 μl ultrapure water 

   1 μl RNAse inhibitor (Thermo Fisher “RiboLock” 10389109) 

   1 μl (200 U/μl) reverse transcriptase (Thermo Fisher “RevertAid M-MuLV” 10161310)

   2 μl 10 mM dNTPs 

5. Incubate at room temperature for 10 min 

6. Incubate at 37° for 1-2h

7. Incubate at 70° for 10 min 

8. You can store the cDNA at -20° indefinitely

9. We will typically take half of each sample (10 μl) to combine to make a neat standard which is further serially diluted 5- fold in TE to give us a range of standards.

10. The remaining sample (10 μl) is diluted 40-fold in TE for the actual assay. This means that a given sample gives enough cDNA to assay hundreds of genes. 

PCR:

Requirements: qPCR mix including SybrGreen, primers

1. Set up reactions on ice. 

2. Mix, per reaction: 

   5 μl 2x qPCR—we use qPCRBIO SyGreen Mix Separate-ROX (PCR Biosystems PB20.14-51)

   0.2 μl 10 μM primer mix 

   4.8 μl DNA template 

3. Primer mix contains 10 μM each forward and reverse gene-specific primers in TE

4. Typically, we set reactions up as follows: If you are measuring expression of Rpl4 in 20 samples, make a mix containing 20x5x1.1 = 110 μl qPCR Mix and 20x0.2x1.1 = 4.4 μl Rpl4 primer mix. (The 1.1-fold correction is again to avoid running out early.) Add 5.2 μl of this mix to each reaction tube. Then add 4.8 μl of DNA template to each tube. 

5. We perform amplification in a RotorGene 6000/RotorGene Q: 

   1x 95° 10 min 

   Then 40-50 cycles: 

   95° 15 sec 

   57° 30 sec 

   72° 30 sec 

3.  Measuring glucose, trehalose and glycogen

This protocol uses a glucose oxidase-based glucose detection reagent designed for clinical chemistry labs. Glucose oxidase reacts with glucose and oxygen to generate peroxide. The peroxide is then measured using a standard chromogenic substrate. The absorbance is measured at 492 nm.

In order to generate a correct measurement, you need to measure a bunch of parameters: the turbidity from the fly, independent of carbohydrates; the absorbance of the glucose reagent without glucose added; and so on. At the end of this protocol, a computation is included that will allow you to extract the part of each reading coming from the carbohydrate being measured.

Measurement of trehalose and glycogen uses enzymes that cut these carbohydrates into individual glucose units (trehalase and amyloglucosidase, respectively). One molecule of trehalose contains two molecules of glucose; one molecule of glycogen can contain hundreds of molecules of glucose. We measure the mass of glucose liberated—we don’t care about the number of molecules of glycogen, what we care about is the mass of glucose it represents.

1. Smash flies on ice in TE + 0.1% Triton X-100 ( We use 25µl/adult male fly)

2. Transfer the samples directly from ice to a heat block at 75° and incubate for 20 minutes to kill endogenous trehalase activity

   If you don’t do this heat treatment, your glucose measurement will also include trehalose, and your trehalose measurement will be around zero, maybe negative.

3. Freeze at -80° until needed

4. Thaw samples at 60° for 5 minutes. Vortex the samples and spin them for 5 minutes at 12.000g at room temperature

   Try not to pipette too many macroscopic chunks of fly into the plate as this will dramatically increase the background

5. Get your Glucose reagent. Make two extra mixes with this: one supplemented with 1/100 vol amyloglucosidase, one supplemented with 1/100 vol trehalase. These will now be glycogen and trehalose reagents, respectively. You need to make these fresh each time; just make as much as you need (you need 200µl of each reagent per sample, plus a bit)

6. You will measure standards with the glucose reagent, but not with the glycogen or trehalose reagents

7. Add 10µl of each sample to four wells in a 96-well plate (i.e., each sample will be measured four times—once for fly background, once for glucose, once for trehalose, once for glycogen)

8. If the following description is confusing, refer to the plate diagram below

9. To the first set of samples add 200µl of water. These are for measuring fly background absorbance

10. To the second set of samples add 200µl of glucose reagent

11. To the third set of samples add 200µl of trehalose reagent. To the fourth set of samples add 200µl of glycogen reagent

    If any of the wells contain macroscopic fly fragments, remove them. If any well has bubbles, pop them. Fly parts and bubbles will skew the reading

12. Incubate the plate at 37° for at least 1 hour. Overnight may be better—trehalase is slow.

13. Read A500 on a plate-reader.

Standards:

1. Standards are glucose reagent (200µl) supplemented with known quantities of glucose.

2. Standard 1 can be 10µl of 10g/l glucose; make a dilution series from this mostconcentrated standard (e.g. ½, ¼, 1/8...)

3. Since your reaction contains 10µl of the standard and 200µl of reagent, your most concentrated standard reaction will contain 100µg glucose, the next standard reaction 50µg, and so on.

   Don’t worry about the most concentrated standards being nonlinear: they have much more carbohydrate than a fly (typical reading from fly samples will never exceed 10µg glucose or so). Just exclude the nonlinear mostconcentrated standards from your calculations

4. It’s important that your standards also include a measurement of the reagent alone, with no added glucose

Calculations:

1. The rows of the plate have letters (A-H). The columns have numbers (1-12). For a fly in column 1, we have measurements A1 (fly+water), B1 (fly+glucose reagent), C1 (fly+trehalose reagent), D1 (fly+glycogen reagent), E1 (water alone), and R (reagent alone— from your standard curve)

2. Then we can get the following measurements:

   Glycogen = D1-B1 Trehalose = C1-B1

   Glucose = B1-(A1-E1)-R = B1-A1+E1-R

   If your measurements give negative numbers here, something is not working properly.

3. You can convert these measurements into µg/fly using linear regression on your standards (remember to subtract R from each standard reading!)

4. Each sample contains 40% of one fly. When you do this linear regression, it is best to force the regression to go through zero, to prevent very small values from registering as negative

5. Remember to exclude the most concentrated standards if they are outside the linear range (it’s easy to tell because each measurement should be about double the one below).

 4. Bacterial quantification by qPCR

 Genomic DNA extraction:

1. Make up lysis buffer: 1x TE, 1% Triton X-100. Add 1/100 vol proteinase K right before use. You need 100μl/fly

2. Smash individual flies in 100μl lysis buffer. Try to do a good job of homogenizing them with the pestle

3. Incubate 3 hours at 55°

4. Incubate 10 minutes at 95°

5. Use 4.8μl of this sample per 10μl qPCR reaction

Making bacterial standards:

1. Make 1ml of a suspension of bacteria in PBS at OD=1.

2. Pellet bacteria by centrifugation at 4000xg for 5 minutes.

3. Resuspend pellet in 250μl lysis buffer.

4. Incubate 3 hours at 55°.

5. Incubate 10 minutes at 95°.

6.  Make a dilution series of this bacterial lysate in TE supplemented with 1/1000 vol glycogen.

   You will likely get poor amplification from the un-diluted sample. For reference, 4.8 μl of undiluted Listeria lysate should contain 2.7x107 copies of the Listeria genome.