Transcript RNA Extraction

 The
HIV virus can mutate in HIV positive
patients taking Anti-Retroviral Therapy (ART)
 Their
HIV strain has now become drug
resistant (DR), and their current medication
no longer works
 This
increases the patient’s viral load, and
creates the possibility of passing on a drug
resistant strain of HIV
 To
create a device which can qualitatively
detect if an HIV positive patient’s ART is
working or if their strain has mutated
 Create
an automated platform which
combines RNA extraction, a PCR
Thermocycler, and Gel Electrophoresis to
give a qualitative result.
 Use
RNA extraction methods to purify RNA
 Send a single strand of RNA to PCR group
 PCR
group turns single strand of RNA to cDNA
(complementary DNA) via RNA transcriptase.
 Then use PCR to amplify DNA segment
 PCR group sends amplified DNA to
electrophoresis group
 Electrophoresis
group runs samples through
gel, sample can then be analyzed.
 If a band is present on a specific segment of
the gel, then HIV is present.
 Can conclude the patient’s HIV strain is drug
resistant, due to the presence of HIV
 Steps




Lyse serum
Adjust binding conditions
Bind to glass mesh
Remove contaminants



of RNA extraction:
Inhibitor Removal Buffer
Wash Buffer
Elute
Roche :
High Pure Viral RNA Kit
 Obtain
sample from RNA team
 Convert RNA to cDNA
 Amplify DNA via PCR

Heat and cool sample according to protocol
 Export
to Gel Electrophoresis team
 Make
Agarose Gel with Ethidium Bromide
 Load amplified DNA from PCR/Thermocycler
into gel
 Run voltage (100V) through gel for
approximately 20-40 minutes
 View gel under UV light to determine
presence of HIV

Visible due to ethidium bromide tag
 Building
our own box based on open source
designs
 Looking for inexpensive, safer alternatives to
ethidium bromide

Create safe waste disposal protocols if ethidium
bromide must be used.
 Designing

This means Resource Limited Settings
 Size

for Developing Nations.
should be reasonable
If it is too large it will be too expensive to ship
 Cost
 Must

be incredibly reliable
Overly complex designs are more likely to break
 Would
like to be able to run between 20-100
patient samples in a day

Blood pooling methods