Modified RNA (MRT) Heike Krber, Dieter Schimkat, Rolf Kaiser MRT - - PowerPoint PPT Presentation

AK_02122003.ppt

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Modified RNA (MRT)

Heike Körber, Dieter Schimkat, Rolf Kaiser

RNA

MRT Protection MRT Deprotection Purification

AK_02122003.ppt

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Analysis MRT-RNA

The mechanism of MRT MRT protection leeds to acetylation of all 2`-OH groups of the RNA molecule

OH OH O O O O O O O O N N NH NH O O O O O O O O N N NH NH M

AK_02122003.ppt

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dilutions of HIV-1 in human plasma from 0 – 50 000 copies / ml were prepared with MRTpellet and purified with HXA-Beads + DyeEx The samples were analysed in COBAS Amplicor

Result:

MRT gives comparable results to UltraSense

target too low 1 10 100 1000 10000 100000 US - 50 000 MRT - 50 000 US - 5000 MRT - 5000 US - 500 MRT - 500 US - 50 MRT - 50 US - 0 MRT - 0 1 10 100 1000 10000 100000 US - MRT - US - MRT - US - MRT - US - 50 MRT - US - MRT -

log copies / ml

HIV COBAS Amplicor + HXA-Beads

test series A test series B test series A test series B

UltraSense protocol MRTpellet protocol

Viral analysis Assays - HIV-1

HIV:

Retrovirus: ss RNA, enveloped

AK_02122003.ppt

4 Experimental setup:

Human samples with 100 – 10 000 copies HIV / ml were prepared with Qiagen and MRT + Silica-Beads The samples were analysed in COBAS TaqMan

Result:

MRT gives comparable results to Qiagen, but for MRT in low concentrations one sample failed and one IC failed

log copies / ml

HIV COBAS TaqMan + Silica-Beads

Qiagen protocol MRT protocol invalid IC

Viral analysis Assays - HIV-1

HIV:

Retrovirus: ss RNA, enveloped

MRT protocol + Silica-Beads

1 10 100 1000 10000 1000000 100000 Qiagen 10 000 MRT 10 000 Qiagen 1 000 MRT 1 000 Qiagen 100 MRT 100

MRT – invalid no target

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Viral analysis Assays - ECHO-12

Overview viral analysis ECHO-12

Light Cycler Assay (Roche)

ECHO-12 analysis in human plasma

HXA: Sensitivity +++ Reproduction +++ Stability +++ (50 days) Snap: Sensitivity +++ Reproduction ++ (Qiagen reference ) Stability ++ (4 weeks)

ECHO-12 analysis in whole blood

Snap: Sensitivity + Non-Infectivity MRT treated viruses are not pathogen

ECHO-12:

Entero virus: ss RNA, non enveloped

Uni Köln

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Viral analysis Assays - ECHO-12

Experimental setup:

Enterovirus was spiked into whole blood with 10000 and 100000 copies / ml from a quantified cell culture stock. The samples were treated with MRT and purified with Silica-Beads. No reference is available! Analysis was carried out in a specific Roche Light Cycler Assay.

Result:

MRT gives lower yields, than the input of virus, but within the same range

1 10 100 1000 10000 100000 1000000 I n p u t 1 . M R T M R T I n p u t 1 . M R T M R T

log copies / ml

ECHO-12 Light Cycler from whole blood

input copies / ml MRT + Silica-Beads

ECHO-12:

Entero virus: ss RNA plus orientated, non enveloped

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7 Experimental setup:

Enterovirus was spiked into plasma with 100 000 copies / ml, the plasma was treated in different ways and stored at RT, the samples were purified at the designated time points. Analysis was carried out in a specific Roche Light Cycler Assay.

Result:

MRT is comparable to Qiagen and RNAlater, lysed virus degrades after

• ne week, the virus is stable in plasma all the time

ECHO-12 Light Cycler Stability Test

⇒ pure plasma without treatment ⇒ treated with Qiagen lysis buffer ⇒ treated with RNAlater ⇒ protected with MRT purified with HXA-Beads + Microcon

Viral analysis Assays - ECHO-12

log copies / ml

1 10 100 1000 10000 100000 1000000 5 10 15 20 25 30 35 40 45 50

days at room temperature

ECHO-12:

Entero virus: ss RNA, non enveloped

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Reference MRT – HXA-Beads

HSV-1 Light Cycler + HXA-Beads

Experimental setup:

200 µl human plasma were spiked with cultivated HSV-1. Samples were treated according to the QIAamp DNA Blood Mini Kit (Qiagen) and with MRT, purified with HXA-Beads + Microcon. Analysis was carried out in a specific Roche Light Cycler Assay.

Result:

MRT gives comparable or better results than the reference method.

HSV:

herpes virus: ds DNA, enveloped

crossing points

Viral analysis Assays - HSV-1

1 3 5 7 9 11

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9 negative control positive control MRT inactivated HSV Qualitative PCRs were carried out from all culture supernatants: negative control shows no amplification positive control shows amplification MRT sample shows amplification (DNA!)‏ Experimental setup:

cultivated human fibroblasts were incubated with supernatant of cultivated human fibroblast, MRT treated negative control supernatant of HSV-1 infected cultivated human fibroblasts positive control supernatant of HSV-1 infected cultivated human fibroblasts, MRT treated sample

Result:

MRT inactivates HSV-1.

MRT + Virus Inaktivierung

non infectivity of primary samples treated with MRT

HSV:

Herpes Virus: ds DNA, enveloped

AK_02122003.ppt

10 negative control positive control MRT inactivated ECHO-12 Qualitative PCRs were carried out from all culture supernatants: negative control shows no amplification positive control shows amplification MRT sample can be amplified after MRT Deprotection protocol Experimental setup:

cultivated green monkey cells were incubated with supernatant of cultivated green monkey cells, MRT treated negative control supernatant of ECHO-12 infected cultivated green monkey cells positive control supernatant of ECHO-12 infected cultivated green monkey cells, MRT treated sample

Result:

MRT inactivates ECHO-12.

MRT + Virus Inaktivierung

non infectivity of primary samples treated with MRT

ECHO-12:

Entero virus: ss RNA plus orientated, non enveloped

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11 p24 ELISA was carried out from all culture supernatants: negative control shows no p24 positive control shows p24 MRT sample shows no p24 negative control positive control MRT inactivated HIV-1 Experimental setup:

cultivated human donor lymphocytes were incubated with supernatant of cultivated human donor lymphocytes, MRT treated negative control supernatant of HIV-1 infected cultivated human donor lymphocytes positive control supernatant of HIV-1 infected cultivated human donor lymphocytes, MRT treated sample

Result:

MRT inactivates HIV.

non infectivity of primary samples treated with MRT

MRT + Virus Inaktivierung HIV:

Retrovirus: ss RNA, enveloped

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Humane Spender Lymphozyten wurden inkubiert mit

MRT behandelten HIV Kulturen ⇒ Spender Lymphozyten zeigen keine Symptome einer HIV Infektion ⇒ p24 ELISA ist negativ

MRT + Virus Inaktivierung

Inaktivierung infektiöser humaner Proben HIV

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HIV1 HIV1 -

• Lymphocytenkultur

Lymphocytenkultur

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Lymphocytenkultur Lymphocytenkultur

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HIV produzierende Lymphozyten