Preparation of the dsRNA solution
1.Clone target sequences of genes into T-easy vector, with length around 500bp. (Caution: Make sure target sequence does not contain the whole length of qRT-PCR sequence)
2.Set up at least a 50 μl PCR reaction to amplify dsRNA synthesis template using forward and reverse primers with T7 RNA polymerase promoter conjugated.
3.Purify the PCR product using QIAEX II Gel Extraction Kit, and dilute in nuclease free water.
4.Measure the template concentration using a spectrophotometer (Nanodrop), and identify the quality by electrophoresis on 1% (wt/vol) agarose gel with DNA marker. Only a single band of right size should be detected (the length of target sequence plus 46 bp of two T7 promoters).
5.Use a total of 200ng DNA template to synthesis dsRNA with MEGAscript T7 transcription kit. (Tip: High content and quality of DNA template could increase dsRNA production.)
6.Incubate reaction at 37 °C for more than 16 h in an air bath. (Critical: Although manufacturer recommend a normal reaction incubate at 37 °C for 2-4 h, we found that a longer time increases dsRNA yield to final concentration of dsRNA as high as 10 μg/μl; Tip: Air bath is better than water bath, in order to maintain the volume of reaction and avoid water condensing on the cap of microcentrifuge tub.)
7.Measure the RNA concentration using a spectrophotometer (Nanodrop) using a 1:10 dilution of dsRNA product in water. (Critical: Dilute dsRNA product could reduce the impact of ions on determination of RNA quality.)
8.Examine 0.5 μl of the dsRNA product using electrophoresis on 1% (wt/vol) agarose gel with DNA marker. dsRNA size should be the same as that of the DNA template.
9.Add 1 μl TURBO DNase, and incubate at 37 °C for 15 min.
10.Terminate the reaction at 65 °C for 10 min.
11.(Optional) Purify dsRNA product via LiCl precipitation, and resuspend in nuclease-free water. (Tip: It is our experience that no difference in the result of RNA interference in planthopper body between purified and unpurified dsRNA. More importantly, LiCl precipitation could result in the loss of a large part of dsRNA product.)
12.Adjust the concentration of dsRNA product to 5 μg/μl.
13.Centrifuge at 12,000 rpm for at least 5 min in 4 °C. (Critical: High speed centrifuge is essential to remove impurities, which is critical prior to microinjection.)
14.Store dsRNA product at -80 °C until use.
Preparation of the agarose plates
1.Dissolve 1-2% (wt/vol) agarose in PBS in a microwave oven.
2.Pour liquid into plastic dishes to almost overflowing. (Tip: Different sizes of dishes should be used for different instar nymphs of BPH: 3cm diameter dishes for 1st and 2nd instar nymphs and 6cm diameter dishes for 3rd, 4th, 5th instar nymphs and adults.)
3.Before the agarose solidifies, place capillary (insect pin or toothpick) gently on the edge of dishes (Figure 1). (Caution: Do not let immerse strips in the liquid; Tip: an insect pin can be used for 3cm diameter dishes, and capillary or toothpick for 6cm diameter dishes; see Figure 1.)
4.Remove capillary (insect pin or toothpick), and immerse agarose plates in PBS at 4 °C until ready to use. (Critical: Keep the groove integrity, which ensure BPH body in the right position.)
Preparation of rearing jar
1.Cut absorbent cotton into 7cm square pieces of sufficient thickness.
2.Each jar needs two squares of cotton. Place one piece on the bottom and infiltrate with rice nutrient fluid. Punch four holes in another cotton square around the center and place 10 fresh rice seedlings of each hole.
3.Place this cotton square with rice into the jar, and compress around the edge of square with forceps (Figure 2). (Caution: As high humidity increase mortality of BPH, jar should not be watered anymore until the BPH are put in; do not leave any gaps at the edge of cotton, most of BPH will die if they jump or fall into the gaps.)
Microinjection of dsRNA into planthopper
1.We are now ready to start microinjection of the dsRNA.
2.Pat dry the agarose plates with paper towels, and place on ice. (Critical: Keep agarose plates dry for convenient microinjection and to decrease mortality.)
3.Anaesthetize BPH using CO2 for 30s (Figure 3a). (Caution: Do not use ice to anaesthetize BPH, otherwise they will die.)
4.Place them on agarose plates, and use a pointed brush to place BPH in the groove (Figure 3b). (Critical: It is best to lay them flat at right angles to the injection needle; Tip: Use only a few brush hairs to place BPH gently, in order to minimize damage to the BPH body.)
5.Pull capillary for microinjection needles using a micropipette puller (P-97, Sutter Instrument) (Figure 3c). A normal program is heat = 800; pull = 150; vel = 150; time = 80. (Tip: Store microinjection needles in 15 ml microcentrifuge tubes, which protects the points of the needles (Figure 3d).)
6.Add 10 μl of dsRNA product in the microinjection needle using microloader (Figure 3e). (Caution: Do not allow air bubbles into microloader and needle; Tip: microloader could use repeatedly.)
7.(Optional) For practicing microinjection, add Blue Dextran solution into dsRNA product to indicate whether the liquid has been successfully injected.
8.Insert microinjection needle into grip head and fix on capillary holder of the microinjection instruments (Figure 3f).
9.Set the injection parameters. A normal program is injection pressure (pi) = 1000 hPa, injection time (ti) = 0.5 s and compensation pressure (pc) = 20 hPa, depending on tip size of needle.
10.Cut microinjection needle using microforceps, and adjust size of needles to be suitable to penetrate BPH body (Figure 3g). A suitable size of the point is very fine but unbending when inject into the body (Figure 3h). (Critical: A beveled point is the most convenient shape for injection, to minimize damage to the integument and avoid bleeding.)
11.Choose the right injection sites. It is our experience that two sites are most suitable for injection: (1) the membrane between the meso- and meta-thoracic legs; (2) the membrane between thorax and abdomen. Both these position are the thinnest places in the body and the least subject to bleeding. Other places can also be used, such as the membrane between abdominal segments and around the coxae of the meso- and meta-thoracic legs. (Caution: Do not insert needle deeply: this increases mortality; Tip: Early emerged (first day) of each instar is a better time point for injection as the body is not yet turgid; once the needle is inserted into the body, immediately pull it out slightly, which avoids tissues blocking the tip of needle.)
12.Inject approximately 5nl, 10nl, 25nl, 50nl and 100nl dsRNA solution into 1st, 2nd, 3rd, 4th and 5th instar nymphs, respectively. A larger volume can be injected into adults.
Rearing the planthoppers after injection
1.Gently pick BPH body out of the groove after injected by a few brush hairs. (Caution: Overexertion could hurt BPH body.)
2.Put them in rearing jar with nylon mesh covered. Each jar contains no more than 150 individuals.
3.Immediately move rearing jar to climate chamber with 26 °C (±0.5 °C), photoperiod of 16:8 h (light : dark) and relative humidity of 50% (±5%).
4.Check rearing jar 2 h after injection, and check whether each of the BPHs revives.
5.Renew fresh rice seedlings every three or four days. (Critical: Good condition of fresh rice seedlings reduces BPH mortality.)
Verification of RNAi
1.Three days after injection, examine the gene silencing efficiency using qRT-PCR. Extract total RNA of 20 individuals of BPH with three replicates.
2.(Optional) Test gene expression at the protein level by western blotting using a specific antibody.