siRNA confirmation

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas. Sed massa ipsum, maximus at dictum dapibus, convallis eget augue. Cras purus mauris, mattis quis ornare a, porttitor non quam. Donec sem felis, feugiat vitae porta sit amet, laoreet a leo. Proin in arcu iaculis, facilisis nisi at, rutrum neque. Nullam condimentum, urna quis pharetra lacinia, justo quam fermentum augue, at porta turpis turpis aliquet risus. Aenean lacinia nunc eu porttitor aliquet. Aenean mattis posuere felis, ac finibus est sodales sit amet. Integer lobortis metus vitae ante sollicitudin pharetra. Quisque egestas sem quis ante tristique cursus. Mauris non blandit velit. Ut euismod ut risus rutrum aliquam.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas.

Sequencing

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas. Sed massa ipsum, maximus at dictum dapibus, convallis eget augue. Cras purus mauris, mattis quis ornare a, porttitor non quam. Donec sem felis, feugiat vitae porta sit amet, laoreet a leo. Proin in arcu iaculis, facilisis nisi at, rutrum neque. Nullam condimentum, urna quis pharetra lacinia, justo quam fermentum augue, at porta turpis turpis aliquet risus. Aenean lacinia nunc eu porttitor aliquet. Aenean mattis posuere felis, ac finibus est sodales sit amet. Integer lobortis metus vitae ante sollicitudin pharetra. Quisque egestas sem quis ante tristique cursus. Mauris non blandit velit. Ut euismod ut risus rutrum aliquam.

Blue BSLA colonies

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas. Sed massa ipsum, maximus at dictum dapibus, convallis eget augue. Cras purus mauris, mattis quis ornare a, porttitor non quam. Donec sem felis, feugiat vitae porta sit amet, laoreet a leo. Proin in arcu iaculis, facilisis nisi at, rutrum neque. Nullam condimentum, urna quis pharetra lacinia, justo quam fermentum augue, at porta turpis turpis aliquet risus. Aenean lacinia nunc eu porttitor aliquet. Aenean mattis posuere felis, ac finibus est sodales sit amet. Integer lobortis metus vitae ante sollicitudin pharetra. Quisque egestas sem quis ante tristique cursus. Mauris non blandit velit. Ut euismod ut risus rutrum aliquam.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas.

Real Time PCR

Since the focus of this project is to verify the effective gene silencing with the four different siRNA we designed, the direct measurement of the amount of mRNA in the cells was required. The first step was to perform a Two-Step Reverse Transcriptase PCR to find out where do the siRNA molecules hybridize with the mRNA. The results are shown below. .

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas.

Table: Expected products for the amplifications in the Two-step RT-PCR.

Product	Expected size
Tubulin	195 bp
Rac I	193 bp
WNT	200 bp
AWD	180 bp
SOD	199 bp

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas. Sed massa ipsum, maximus at dictum dapibus, convallis eget augue. Cras purus mauris, mattis quis ornare a, porttitor non quam. Donec sem felis, feugiat vitae porta sit amet, laoreet a leo. Proin in arcu iaculis, facilisis nisi at, rutrum neque. Nullam condimentum, urna quis pharetra lacinia, justo quam fermentum augue, at porta turpis turpis aliquet risus. Aenean lacinia nunc eu porttitor aliquet. Aenean mattis posuere felis, ac finibus est sodales sit amet. Integer lobortis metus vitae ante sollicitudin pharetra. Quisque egestas sem quis ante tristique cursus. Mauris non blandit velit. Ut euismod ut risus rutrum aliquam.

Diaphorina citri primary cell culture

The main objetive was to develop a primary culture of Diaphorina citri cells, to be transfected with our siRNA and analyzed through flow cytometry. The transfected siRNA sequences were tagged with Alexafluor so they would be visible. This protocol was repeated several times under different conditions, and different compositions of the culture medium were used, which can be seen below. The cells that were cultured in Medium A were able to survive after twelve hours, and replicated at a slow rate. Because of this, the flow cytometry was not performed.

Reagent	Medium A	Medium B
200 mM L-Glutamine	1 mL	1 mL
Penicillin and Streptomycin 1000X	1.5 mL	1.5 mL
Fungizone 500X	0.2 mL	0.2 mL
Schneider Medium	72 mL	80 mL
199 Medium 10X with Hanks’ salts	6 mL	-----
Fetal Bovine Serum (heat-inactivated)	20 mL	20 mL
0.06 M Histidine solution	1 mL	-----

Figure 1. Cell culture 12 hours after extraction.

Figure 2. Cell culture 2 days after extraction, observed under an inverted microscope at 10x. No significant cell replication can be observed compared to the first day,

Figure 3. Cell culture 2 weeks after extraction, observed under an inverted microscope at 10x. Cell replication can be observed.

Diaphorina citri laboratory development

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus efficitur dolor erat, vel lobortis augue mattis nec. Ut sit amet placerat massa. Sed dignissim ante eget nibh sollicitudin, at tincidunt mi fermentum. Curabitur tempus nibh in velit maximus egestas. Sed massa ipsum, maximus at dictum dapibus, convallis eget augue. Cras purus mauris, mattis quis ornare a, porttitor non quam. Donec sem felis, feugiat vitae porta sit amet, laoreet a leo. Proin in arcu iaculis, facilisis nisi at, rutrum neque. Nullam condimentum, urna quis pharetra lacinia, justo quam fermentum augue, at porta turpis turpis aliquet risus. Aenean lacinia nunc eu porttitor aliquet. Aenean mattis posuere felis, ac finibus est sodales sit amet. Integer lobortis metus vitae ante sollicitudin pharetra. Quisque egestas sem quis ante tristique cursus. Mauris non blandit velit. Ut euismod ut risus rutrum aliquam.

Figure 1. Detail of mesh lining the interior of the cages. The size of the mesh prevents the insects from escaping while providing light, air flow and visibility.