Molecular Testing Core

 

Mission

Pamela Milani is the lead of the Molecular Testing Core at Vitalant Research Institute (VRI). Dr Milani is dedicated to advancing molecular technologies and their applications in research and clinical settings; throughout her career, she has specialized in developing and validating genomic, transcriptomic and epigenomic methods based on Next Generation Sequencing (NGS), as well as non-NGS molecular assays such as qPCR and digital PCR. Before joining VRI, Dr Milani led the Genomic Biomarker Group at Bristol Myers Squibb (BMS), where she directed the adaptation, optimization and execution of genomic assays including multi-modal single cell sequencing and targeted NGS. She also developed multiplex digital PCR assays for variant detection and qPCR for oncovirus testing. Before BMS, she served as Senior Manager at Personalis, Inc, where she worked on launching novel genomic platforms for diagnostic applications and multidimensional biomarker identification. She completed her post-doctoral training in biological engineering at the Massachusetts Institute of Technology where she gained expertise in epigenomics, functional genomics and systems biology.

 

Pamela-Milani-VRI-Photo-vers2.jpg
Director: Pamela Milani, PhD.



Pamela Milani, PhD.
360 Spear Street, Suite 200
San Francisco, CA 94105
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DNA and RNA extraction and quantification

Nucleic acid extraction is performed on a variety of sample types including fresh and frozen whole blood, dried blood spots, purified cells, and cell-free body fluids. Manual extraction and enrichment using commercial kits or in-house developed protocols are available. For example, we have optimized protocols for extraction of Trypanosoma cruzi parasite DNA from blood using guanidine HCl lysis, HemoBind™ buffer and target capture; Babesia microti parasite DNA from blood using freeze-thaw lysis and differential centrifugation; and genomic DNA from leukocytes using proteinase K lysis. Automated nucleic acid extraction is available using the Promega Maxwell® 16 System. Quantification and quality control can be done by spectrophotometry using NanoDrop™, by electrophoresis using Agilent 2100 BioAnalyzer, by fluorescence using PicoGreen®, or by qPCR using in-house validated assays.

Real-time PCR

One-step real-time RT-PCR is available using commercial kits. For two-step real-time RT-PCR, we have developed protocols for cDNA synthesis followed by amplification using in-house optimized buffers. We perform qPCR in 96-well or 384-well formats using two LightCycler 480 Systems (Roche) and one 7500 Real-Time PCR System (Applied BioSystems). The Molecular Transfusion Core has designed hundreds of SYBR Green and TaqMan probe based assays for viral, parasitic, human, murine, and primate gene targets. Custom primer/probe assay design can also be performed for gene expression, SNP genotyping, and multiplex detection.

Data analysis

Results are reported based on raw Ct values, quantification relative to standard curves, or relative quantification based on 2^-dCt method. We provide assistance with generating figures and slides and writing text for manuscripts and presentations.

Lani Montalvo

Lani Montalvo
Research Associate III

Daniel Chafets

Daniel Chafets
Research Associate III

Li Wen

Li Wen
Research Associate III

Sonia Bakkour, MD, PhD

Blood Systems Research Institute
360 Spear Street, Suite 200
San Francisco, CA 94105

Phone: (415) 923-5771 ext. 330

DNA and RNA extraction and quantification

Nucleic acid extraction is performed on a variety of sample types including fresh and frozen whole blood, dried blood spots, purified cells, and cell-free body fluids. Manual extraction and enrichment using commercial kits or in-house developed protocols are available. For example, we have optimized protocols for extraction of Trypanosoma cruzi parasite DNA from blood using guanidine HCl lysis, HemoBind™ buffer and target capture; Babesia microti parasite DNA from blood using freeze-thaw lysis and differential centrifugation; and genomic DNA from leukocytes using proteinase K lysis. Automated nucleic acid extraction is available using the Promega Maxwell® 16 System. Quantification and quality control can be done by spectrophotometry using NanoDrop™, by electrophoresis using Agilent 2100 BioAnalyzer, by fluorescence using PicoGreen®, or by qPCR using in-house validated assays.

Real-time PCR

One-step real-time RT-PCR is available using commercial kits. For two-step real-time RT-PCR, we have developed protocols for cDNA synthesis followed by amplification using in-house optimized buffers. We perform qPCR in 96-well or 384-well formats using two LightCycler 480 Systems (Roche) and one 7500 Real-Time PCR System (Applied BioSystems). The Molecular Transfusion Core has designed hundreds of SYBR Green and TaqMan probe based assays for viral, parasitic, human, murine, and primate gene targets. Custom primer/probe assay design can also be performed for gene expression, SNP genotyping, and multiplex detection.

Data analysis

Results are reported based on raw Ct values, quantification relative to standard curves, or relative quantification based on 2^-dCt method. We provide assistance with generating figures and slides and writing text for manuscripts and presentations.

Lani Montalvo

Lani Montalvo
Research Associate III

Daniel Chafets

Daniel Chafets
Research Associate III

Li Wen

Li Wen
Research Associate III

Research Interests

 

The Molecular Transfusion Medicine Core research interests center around the design and optimization of real-time PCR assays for the detection and quantification of viral and parasitic infectious diseases that are of concern to the blood transfusion community. Another major area of focus is the detection of genetic variants that are relevant to blood donation or response to transfusion. We have a long-standing interest in detection of small numbers of donor cells within a recipient (known as microchimerism).

Pathogen Detection

Nucleic acid amplification testing (NAT) has been increasingly adopted to maintain the safety of the blood supply. We have developed real-time PCR assays specific for transfusion-transmitted viruses and parasites. These assays are used in research studies that aim to define prevalence and duration of infection in blood donors and recipients, as well as blood compartmentalization. We also employ our assays as supplemental tests for cross-validation of commercial NAT and serology assays in development.

Microchimerism

Microchimerism is the presence of small populations of genetically disparate cells within a host as a result of transfusion or transplacental trafficking during pregnancy, and may influence immune tolerance in the recipient. We have developed sensitive allele-specific PCR assays that allow the detection and quantification of donor genetic material within recipient cells. These assays have been used to measure transfusion-associated and pregnancy-associated microchimerism in humans, primates and mice.

Damage Associated Molecular Patterns

Cell or tissue injury leads to the release of damage-associated molecular patterns (DAMPs), which stimulate innate immune responses. Our group has developed PCR assays to measure mitochondrial DNA (mtDNA), which is a DAMP that signals through TLR9. Using this assay, we measured mtDNA in plasma of HIV-infected patients, and in blood components. We also developed PCR inhibition assays targeting mtDNA to assess the efficacy of pathogen reduction technologies that induce nucleic acid damage.

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